Systems and methods for the detection of infectious diseases

ABSTRACT

The present invention relates to method of detecting and characterizing one or more  Borrelia  species causing Lyme Disease or tick-borne relapsing fever within a sample from a subject, the method comprising: a) subjecting DNA and/or RNA from the sample to a PCR amplification reaction using primer pairs targeting at least one region of  Borrelia  16S rRNA and at least one region of flaB, ospA, ospB, ospC, glpQ, 16S-23S intergenic spacer (IGS1), 5S-23S intergenic spacer (IGS2), bbk32, dbpA, dbpB, and/or p66; and b) analyzing amplification products resulting from the PCR amplification reaction to detect the one or more  Borrelia  species.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No. 16/076,608, filed Aug. 8, 2018 (published as US20190040455), which is the U.S. National Stage of International Patent Application No. PCT/US2017/017573, filed Feb. 11, 2017, which claims priority to U.S. Provisional Patent Application No. 62/293,873, filed Feb. 11, 2016, the contents of which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII-formatted sequence listing with a file named “91482_201_Sequence_Listing.txt” created on Feb. 9, 2017, and having a size of 85 kilobytes, and is filed concurrently with the specification. The sequence listing contained in this ASCII-formatted document is part of the specification and is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to the field of detection of Borrelia species that cause Lyme Disease and tick-borne relapsing fever in samples from a subject.

BACKGROUND

Lyme disease, also known as Lyme borreliosis, is caused by infection with the bacterial spirochete Borrelia burgdorferi, which is transmitted by the bite of Ixodes ticks. Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii cause Lyme disease in Eurasia and Borrelia burgdorferi and Borrelia mayonii cause Lyme disease in the United States and Canada. B. garinii has been found in pelagic bird colonies off the coast of North America, so there may be potential for infection by this agent in North America. The four Lyme disease agents Borrelia burgdorferi, Borrelia mayonii, Borrelia garinii and Borrelia afzelii are referred to as Borrelia burgdorferi sensu lato, that is, “in the broad sense.” The North American genospecies Borrelia burgdorferi is called Borrelia burgdorferi sensu stricto, “in the strict sense.”

Lyme disease is characterized by three stages: 1) early localized Lyme disease; 2) early disseminated Lyme disease; and 3) late disseminated Lyme disease. A subject may be suspected of having Lyme disease where symptoms are consistent with those of Lyme disease and where an Ixodes tick bite is known or may have occurred. A characteristic rash called erythema migrans occurs in 70-80% of Lyme disease patients at the site of an infected tick bite.

Early localized Lyme disease is characterized by erythema migrans. Early disseminated Lyme disease typically occurs days to weeks after the initial bite by an infected tick and possible signs include secondary erythema migrans, early neuroborreliosis (cranial nerve palsy, meningitis, or radiculoneuropathy) or, uncommonly, Lyme carditis (atrioventricular node conduction block). Non-specific symptoms such as malaise, fever, headache, and muscle and joint pains may be present. Late disseminated Lyme disease occurs months to years after the initial bite by an infected tick. The most common manifestation of late disseminated Lyme disease in North America is Lyme arthritis, which is characterized by intermittent attacks in large joints, particularly the knees. Rarely, late neuroborreliosis develops, with manifestations including encephalopathy, encephalomyelitis, and/or peripheral neuropathy. Wormser, G. P., et al. Clin Infect Dis 2006; 43:1089-1134.

Lyme arthritis is a late manifestation of Lyme disease affecting up to 60% of untreated patients in the United States. Ten percent of patients treated with antibiotics continue to suffer from recurrent bouts of Lyme arthritis, Steere, A. C. and L. Glickstein, Nat Rev Immunol, 2004. 4(2): p. 143-52. Cartilage loss and subsequent bone destruction which are features of osteoarthritis and rheumatoid arthritis also occur in advanced cases of Lyme arthritis, Lawson, J. P. et al., Radiology, 1985, 154(1):37-43. Lyme arthritis develops when the bacteria invade joint tissue, most commonly the knee, and trigger inflammation as part of a strong host immune response. Despite this vigorous immune response, Borrelia are able to persist in joints which are thought to be a protective niche for the bacteria due to limited perfusion, Liang, F. T., et al., Am J Pathol, 2004, 165(3):977-85.

The detection and management of the disease is complicated by several factors, limiting the ability of clinical medicine to rapidly identify patients and subsequently employ appropriate therapy. Important complicating factors in the diagnosis of Lyme borreliosis infection include:

-   -   1. Co-infection: Ixodes ticks may transmit multiple pathogens         while taking a blood meal, which may result in co-infection and         confounding symptoms and test results;     -   2. Unspecific testing: multiple Borrelia species are now known         and other unknown Borrelias likely exist, all of which may cause         false positives on Lyme disease diagnostic tests; and     -   3. Limited sensitivity: Borrelia infections result in typically         low-level bacteremia, and therefore limited target material may         be present in clinical samples.         Another complicating factor is the difficulty of detecting         active infection with the causative agent of Lyme disease in         cases where symptoms are present long after potential exposure         to infected ticks. There is a continuing need for compositions         and methods for the diagnosis of Lyme disease that address these         challenges to rapid detection and treatment.

SUMMARY

The present invention is directed to a method of detecting one or more Borrelia species causing Lyme Disease or tick-borne relapsing fever (TBRF) within a sample from a subject, the method comprising: a) subjecting DNA and/or RNA from the sample to a PCR amplification reaction using primer pairs targeting at least one region of Borrelia 16S rRNA and at least one region of flaB, ospA, ospB, ospC, glpQ, 16S-23S intergenic spacer (IGS1), 5S-23S intergenic spacer (IGS2), bbk32, dbpA, dbpB, and/or p66; and b) analyzing amplification products resulting from the PCR amplification reaction to detect the one or more Borrelia species.

In certain aspects, the primer pairs targeting at least one region of Borrelia 16S rRNA contain sequences selected from the group consisting of SEQ ID NOS: 1-10. In other aspects, RNA from the sample is subject to the PCR amplification reaction with the primer pairs targeting at least one region of Borrelia 16S rRNA.

In yet other aspects, the primer pairs targeting at least one region of flaB, ospA, ospB, ospC, glpQ, 16S-23S intergenic spacer (IGS), 5S-23S intergenic spacer (IGS2), bbk32, dbpA, dbpB, and/or p66 contain sequences selected from the group consisting of SEQ ID NOS: 11-48, SEQ ID NOS: 60-77, SEQ ID NOS: 97-100, and SEQ ID NOS: 219-293.

In one embodiment, the PCR amplification reaction is a multiplex amplification reaction. In another embodiment, the amplification products are analyzed by size determination with agarose gel electrophoresis.

In some embodiments, the amplification products are analyzed by next-generation sequencing (NGS) to determine the sequence of each amplification product. In one embodiment, the primer pairs comprise a universal tail sequence.

In certain aspects, the sequence of each amplification product is mapped to a reference library of known Borrelia sequences to detect the one or more Borrelia species. In other aspects, the one or more Borrelia species are selected from the group consisting of Borrelia afzelii, Borrelia americana, Borrelia andersonii, Borrelia anserina, Borrelia baltazardii, Borrelia bavariensis, Borrelia bissettii, Borrelia brasiliensis, Borrelia burgdorferi, Borrelia californiensis, Borrelia carolinensis, Borrelia caucasica, Borrelia coriaceae, Borrelia crocidurae, Borrelia dugesii, Borrelia duttonii, Borrelia garinii, Borrelia graingeri, Borrelia harveyi, Borrelia hermsii, Borrelia hispanica, Borrelia japonica, Borrelia kurtenbachii, Borrelia latyschewii, Borrelia lonestari, Borrelia lusitaniae, Borrelia mayonii, Borrelia mazzottii, Borrelia merionesi, Borrelia microti, Borrelia miyamotoi, Borrelia parkeri, Borrelia persica, Borrelia queenslandica, Borrelia recurrentis, Borrelia sinica, Borrelia spielmanii, Borrelia tanukii, Borrelia theileri, Borrelia tillae, Borrelia turcica, Borrelia turdi, Borrelia turicatae, Borrelia valaisiana, Borrelia venezuelensis, Borrelia vincentii, and Candidatus Borrelia texasensis. In one aspect, the one or more Borrelia species are Borrelia burgdorferi, Borrelia garinii, Borrelia mayonii, and/or Borrelia afzelii.

In some embodiments, the method further comprises detecting in the sample a Babesia species, an Ehrlichia species, a Bartonella species, Francisella tularensis, Yersinia pestis, Staphylococcus aureus, Anaplasma phagocytophilum, Enterovirus, Powassan and deer tick virus, Rickettsia species, and/or Influenza by subjecting DNA and/or RNA from the sample to a PCR amplification reaction using primer pairs containing sequences selected from the group consisting of SEQ ID NOS: 49-59, SEQ ID NOS: 78-96, SEQ ID NOS: 105-108, and SEQ ID NOS: 294-314.

In other aspects, the sample is whole blood, serum, plasma, buffy coat or connective tissue.

In some embodiments, the subject is an animal. In one embodiment, the animal is a human. In another embodiment, the template is RNA.

In some embodiments, the present invention is directed to a kit for detection of one or more Borrelia species causing Lyme Disease or TBRF, the kit comprising: primer pairs targeting at least one region of Borrelia 16S rRNA and at least one region of flaB, ospA, ospB, ospC, glpQ, 16S-23S intergenic spacer (IGS1), 5S-23S intergenic spacer (IGS2), bbk32, dbpA, dbpB, and/or p66.

In one embodiment, the primer pairs in the kit targeting at least one region of Borrelia 16S rRNA contain sequences selected from the group consisting of SEQ ID NOS: 1-10.

In certain aspects, the primer pairs in the kit targeting at least one region of flaB, ospA, ospB, ospC, glpQ, 16S-23S intergenic spacer (IGS), 5S-23S intergenic spacer (IGS2), bbk32, dbpA, dbpB, and/or p66 contain sequences selected from the group consisting of SEQ ID NOS: 11-48, SEQ ID NOS: 60-77, SEQ ID NOS: 97-100, and SEQ ID NOS: 219-293.

In other aspects, the kit further comprises primer pairs containing sequences selected from the group consisting of SEQ ID NOS: 49-59, SEQ ID NOS: 78-96, SEQ ID NOS: 105-108, and SEQ ID NOS: 294-314.for detecting a Babesia species, an Ehrlichia species, a Bartonella species, Francisella tularensis, Yersinia pestis, Staphylococcus aureus, Anaplasma phagocytophilum, Enterovirus, Powassan and deer tick virus, Rickettsia species, and/or Influenza.

In yet other aspects, the primer pairs in the kit comprise a universal tail sequence. In one aspect, the kit further comprises a nucleotide polymerase, buffer, diluent, and/or excipient.

In one aspect, the kit further comprises one or more primers comprising a sequence selected from SEQ ID NOS: 109 and 110 for amplifying human GAPDH as an internal control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a workflow for the LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay.

FIG. 2 depicts a configuration of multiplex assays used with the LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay to rapidly and accurately diagnose Lyme Disease.

FIG. 3 depicts the Borrelia flaB gene tree with the Borrelia species in the Borrelia burdorferi sensu lato group clustering together and the Borrelia species in the tick-borne relapsing fever (TBRF) species group clustering together.

FIG. 4 depicts mapping of flaB sequence reads against 482 unique DNA sequences to identify Borrelia species that cause Lyme Disease and Borrelia species that do not cause Lyme Disease. For example, the sequence ATGGCCCTATCAT (SEQ ID NO: 476) is specific to Borrelia burdorferi while the sequence ATGGCTTTATAAT (SEQ ID NO: 477) is specific to Borrelia hersmii.

FIG. 5 depicts the Borrelia 16S rDNA tree with the Borrelia species in the Borrelia burdorferi sensu lato group clustering together and the Borrelia species in the tick-borne relapsing fever (TBRF) species group clustering together.

FIG. 6 depicts mapping of Borrelia 16S rDNA sequence reads against 185 unique DNA sequences to identify Borrelia species that cause Lyme Disease and Borrelia species that do not cause Lyme Disease. The arrows depict forward and reverse primers that produce amplicons covering the majority of the Borrelia 16S rDNA sequence.

FIG. 7 depicts colony forming units (CFU) of Borrelia burgdorferi in spiked blood samples plotted against the number of sequence reads for 16S rRNA, flaB-1, flaB-2, and ospB after analysis of either extracted RNA or extracted DNA from the samples. Trendlines are indicated with solid or dashed lines.

DETAILED DESCRIPTION

The present invention provides a method of detecting and characterizing one or more Borrelia species causing Lyme Disease or TBRF within a sample from a subject and addresses the challenges of co-infection that may confound test results, unspecific testing causing false positives on Lyme disease diagnostic tests, and the limited sensitivity available with other methods of detection.

The present invention overcomes these challenges by providing a method A method of detecting one or more Borrelia species causing Lyme Disease or tick-borne relapsing fever (TBRF) within a sample from a subject, the method comprising: a) subjecting DNA and/or RNA from the sample to a PCR amplification reaction using primer pairs targeting at least one region specific to the Borrelia genus, at least one region specific to Borrelia burgdorferi, and/or at least one non-Lyme Borrelia spp. region; and b) analyzing amplification products resulting from the PCR amplification reaction to detect the one or more Borrelia species.

In some embodiments, the primer pairs of the present invention target at least one region of an outer surface protein gene of Borrelia burgdorferi. The Borrelia burgdorferi outer surface proteins include ospA, ospB, ospD, ospC, bba64, ospF, bbk32, dbpA, dbpB, and vlsE. Borrelia burgdorferi outer surface proteins play role in persistence within ticks (ospA, ospB, ospD), mammalian host transmission (ospC, bba64), host cell adhesion (ospF, bbk32, dbpA, dbpB), and in evasion of the host immune system (vlsE). OspC triggers innate immune system via signaling through TLR1, TLR2 and TLR6 receptors. See Oosting, Marije et al. (2016) “Innate immunity networks during infection with Borrelia burgdorferi,” Critical Reviews in Microbiology 42 (2): 233-244.

In certain aspects, the primer pairs of the present invention target at least one region of an intergenic spacer (IGS) region. An IGS region is a region of non-coding DNA between genes and includes the spacer DNA between the many tandemly repeated copies of the ribosomal RNA genes. In one aspect, the IGS region is the region between the 16S and the 23S genes (i.e., 16S-23S intergenic spacer (IGS1)) and/or the region between the 5S and the 23S genes (i.e., 5S-23S intergenic spacer (IGS2)).

In other aspects, the primer pairs of the present invention target at least one region of a porin gene in Borrelia burgdorferi. In some embodiments, the porin gene is selected from the group consisting of p66, p13 and oms28. In one aspect, the porin gene is p66.

In yet other aspects, the primer pairs of the present invention target at least one region of a glycerophosphodiester phosphodiesterase gene (glpQ) from Borrelia spp.

In some embodiments, the primer pairs of the present invention target at least one region of ospA, ospC, CRASP (complement regulator-acquiring surface protein) including CRASP-1 (cspA), CRASP-2 (cspZ), CRASP-3 (erpP), CRASP-4 (erpC), CRASP-5 (erpA), Erp (OspEF-related protein) A, C, and P, bbk32, dbp (decorin-binding proteins) A and B, bgp (Borrelia glycosaminoglycan-binding protein), revA, revB, bb0347, erpX, p66, bbb07, ospC, vlsE, lmp1, and/or ospF family (ospF and G, erpK and L). See Coburn, J., et al. (2013) “Illuminating the roles of the Borrelia burgdorferi adhesins,” Trends in Microbiology, 21(8), 372-379.

As used herein, “amplification reaction” refers to a method of detecting target nucleic acid by in vitro amplification of DNA or RNA.

As used herein, “polymerase chain reaction (PCR)” refers to the amplification of a specific DNA sequence, termed target or template sequence, that is present in a mixture, by adding two or more short oligonucleotides, also called primers, that are specific for the terminal or outer limits of the template sequence. The template-primers mixture is subjected to repeated cycles of heating to separate (melt) the double-stranded DNA and cooling in the presence of nucleotides and DNA polymerase such that the template sequence is copied at each cycle.

The term “primer” refers to DNA oligonucleotides complementary to a region of DNA and serves as the initiation of amplification reaction from the 5′ to 3′ direction.

The term “primer pair” refers to the forward and reverse primers in an amplification reaction leading to amplification of a double-stranded DNA region of the target.

The term “target” refers to a nucleic acid region bound by a primer pair that is amplified through an amplification reaction. The PCR “product” or “amplicon” is the amplified nucleic acid resulting from PCR of a set of primer pairs.

The term “multiplex amplification reaction” herein refers to the detection of more than one template in a mixture by the addition of more than one set of oligonucleotide primers.

As described in greater detail herein, some embodiments of the invention may include amplicon-based sequencing of the one or more markers to make the aforementioned determinations. Some embodiments of the invention include systems and methods of preparing samples for one or more downstream processes that can be used for assessing one or more markers for any of the previously mentioned purposes. Some embodiments of the invention may comprise a universal indexing sequencing strategy for use in downstream sequencing platform processes. By way of example only, some embodiments of the invention comprise a universal indexing sequencing strategy that can be used to amplify multiple genomic regions (e.g., markers, as described below) from a DNA sample simultaneously in a single reaction for the sequencing of one or more amplicons. One or more embodiments of the invention can be used with any desired sequencing platform, such as the ILLUMINA® Next Generation Sequencing (e.g., MiSEQ) platform, Life Technologies' Ion Torrent System, or any other sequencing system now known or developed in the future.

Some embodiments may be configured to enable relatively simple, rapid (e.g., microorganism-culture independent), inexpensive, and efficient preparation of samples for use on, in, and/or with downstream sequencing platforms. For example, some embodiments may use a sequence coupled to one or more oligonucleotides/primers (as used herein, oligonucleotides and primers are used interchangeably). More specifically, one or more amplicons per sample can be generated using a hybrid oligonucleotide that is designed for amplification of a marker and incorporation of at least one universal tail sequence into the resulting amplicon. As a result, additional steps that may be conventionally required to prepare samples for sequencing can be limited or removed entirely. Further information regarding the universal tail, amplicon-based sequencing strategy can be found in PCT/US2014/064890, which is hereby incorporated by reference in its entirety for all purposes.

In some embodiments, the methodology may include performing downstream sequencing on one or more amplicons. For example, in order to minimize and/or eliminate the need for cultures of microorganisms or large inputs of nucleic acids, methodologies of the instant invention may include an initial PCR step to create amplicons that correspond to the one or more pre-selected markers. As such, some embodiments require only limited amounts of starting material are necessary and the starting material need not be of high quality (e.g., genomic DNA, crude DNA extracts, single stranded DNA, RNA, cDNA, etc.). In contrast, many conventional sample preparation systems may require relatively large amounts of starting material of relatively high quality, which can limit the use of some conventional systems.

Some embodiments of the invention can be used for and/or in complement with high-throughput amplicon sequencing of markers, which can be very useful for a variety of molecular genetic genotyping/predicted-phenotyping applications, including clinical sample analysis. For example, use of the systems and methods of the invention can be employed with sequencing platforms to provide rapid, high-yield sequence data, which can enable the sequencing of multiple markers/amplicons from many samples in a relatively short period of time. Specifically, in some embodiments, amplicons can be selected and PCR reactions can be designed to provide information that can be used to make clinically relevant determinations after sequencing of the amplicons.

In some preferred aspects, the methodology may include creating a series of oligonucleotides designed to provide multiplexed amplification of one or more markers to produce the desired amplicons. In particular, the one or more markers and amplicons thereof can be selected/amplified to provide users with clinically relevant information related to identification of one or more potentially infectious microorganisms and/or viruses and phenotypic and genotypic information about the microorganisms and/or viruses (e.g., Borrelia strain identity and 16S-23S intergenic spacer (IGS) sequence variance). After production of the amplicons (e.g., via PCR amplification), which may include the universal tail sequences, the method may include processing the resulting amplicons for downstream sequencing and thereafter sequencing the processed amplicons. After processing and analysis of the resulting sequencing data, one of skill in the art can make any necessary determinations regarding the identification of one or more microorganisms and/or viruses that may have been contained within the sample and predicted-phenotypic and/or genotypic information revealed.

Generally, some embodiments of the present invention can be used to detect, identify, assess, sequence, or otherwise evaluate a marker. A marker may be any molecular structure produced by a cell, expressed inside the cell, accessible on the cell surface or secreted by the cell. A marker may be any protein, carbohydrate, fatty acid, nucleic acid, catalytic site, or any combination of these such as an enzyme, glycoprotein, cell membrane, virus, a particular cell, or other uni- or multimolecular structure. A marker may be represented by a sequence of a nucleic acid or any other molecules derived from the nucleic acid. Examples of such nucleic acids include miRNA, tRNA, siRNA, mRNA, cDNA, genomic DNA sequences, single-stranded DNA, or complementary sequences thereof. Alternatively, a marker may be represented by a protein sequence. The concept of a marker is not limited to the exact nucleic acid sequence or protein sequence or products thereof; rather it encompasses all molecules that may be detected by a method of assessing the marker. Without being limited by the theory, the detection, identification, assessment, sequencing, or any other evaluation of the marker may encompass an assessment of a change in copy number (e.g., copy number of a gene or other forms of nucleic acid) or in the detection of one or more translocations. Moreover, in some embodiments, the marker may be relevant to a particular phenotype or genotype. By way of example only, in some embodiments, the marker may be related to phenotypes including antibiotic resistance, virulence, or any other phenotype.

Therefore, examples of molecules encompassed by a marker represented by a particular sequence further include alleles of the gene used as a marker. An allele includes any form of a particular nucleic acid that may be recognized as a form of the particular nucleic acid on account of its location, sequence, or any other characteristic that may identify it as being a form of the particular gene. Alleles include but need not be limited to forms of a gene that include point mutations, silent mutations, deletions, frameshift mutations, single nucleotide polymorphisms (SNPs), inversions, translocations, heterochromatic insertions, and differentially methylated sequences relative to a reference gene, whether alone or in combination. An allele of a gene may or may not produce a functional protein; may produce a protein with altered function, localization, stability, dimerization, or protein-protein interaction; may have overexpression, underexpression or no expression; may have altered temporal or spatial expression specificity; or may have altered copy number (e.g., greater or less numbers of copies of the allele). An allele may also be called a mutation or a mutant. An allele may be compared to another allele that may be termed a wild type form of an allele. In some cases, the wild type allele is more common than the mutant.

In some aspects, the markers may include one or more sets of amplifiable nucleic acids that can provide diagnostic information about the microorganisms and/or viruses. For example, the markers may include amplifiable nucleic acid sequences that can be used to assess the presence and/or absence of one or more microorganism and/or virus that may have the potential to cause a diseased state in the subject. In some embodiments, the markers may include amplifiable nucleic acid sequences that can be used to identify one or more of the following exemplary microorganisms and/or viruses: Borrelia spp. (including but not limited to Borrelia afzelii, Borrelia americana, Borrelia andersonii, Borrelia anserina, Borrelia baltazardii, Borrelia bavariensis, Borrelia bissettii, Borrelia brasiliensis, Borrelia burgdorferi, Borrelia californiensis, Borrelia carolinensis, Borrelia caucasica, Borrelia coriaceae, Borrelia crocidurae, Borrelia dugesii, Borrelia duttonii, Borrelia garinii, Borrelia graingeri, Borrelia harveyi, Borrelia hermsii, Borrelia hispanica, Borrelia japonica, Borrelia kurtenbachii, Borrelia latyschewii, Borrelia lonestari, Borrelia lusitaniae, Borrelia mayonii, Borrelia mazzottii, Borrelia merionesi, Borrelia microti, Borrelia miyamotoi, Borrelia parkeri, Borrelia persica, Borrelia queenslandica, Borrelia recurrentis, Borrelia sinica, Borrelia spielmanii, Borrelia tanukii, Borrelia theileri, Borrelia tillae, Borrelia turcica, Borrelia turdi, Borrelia turicatae, Borrelia valaisiana, Borrelia venezuelensis, Borrelia vincentii, and Candidatus Borrelia texasensis), Anaplasma phagocytophilum, Ehrlichia spp., Staphylococcus aureus, Yersinia pestis, Francisella tularensis, Bartonella spp., Babesia spp., Influenza virus, and Enterovirus.

In some embodiments, the methods may include the use of one or more than one marker per microorganism or virus. Moreover, in some embodiments, one or more of the microorganisms and/or viruses may not be considered pathogenic to certain subjects, but the methodology employed herein can still rely on detection of pathogenic and non-pathogenic microorganisms and/or viruses for differential diagnoses/diagnostics. In some embodiments, the oligonucleotides (with or without the universal tail sequences detailed herein) listed in Table 1, Table 2, and Table 3 can be used with embodiments of the invention to amplify one or more markers from the microorganisms and/or viruses to provide diagnostic/identification information to the user.

Moreover, in some embodiments, one or more the markers associated with the plurality of microorganisms and/or viruses can be amplified in a multiplex manner. For example, in some aspects, nucleic acids can be obtained from the sample and the oligonucleotides used to amplify one or more of the markers used to identify/diagnose can be added to a single mixture to produce a plurality of amplicons in a single reaction mixture. In other aspects, the oligonucleotides can be added to multiple mixtures to provide for the creation of multiple amplicons in multiple mixtures.

Moreover, in some embodiments, one or more the markers can be amplified in a multiplex manner. For example, in some aspects, nucleic acids can be obtained from the sample and the oligonucleotides used to amplify one or more of the markers used to identify the strain of the microorganism or virus can be added to a single mixture to produce a plurality of amplicons in a single reaction mixture. In other aspects, the oligonucleotides can be added to multiple mixtures to provide for the creation of multiple amplicons in multiple mixtures. In some aspects, amplification of the markers used to identify microorganisms and/or viruses/diagnose an infection can also occur in a multiplex manner such that some or all of the amplicons are generated in a single reaction for a particular sample. In other aspects, amplification of the markers used to identify microorganisms and/or viruses/diagnose an infection can occur in multiple reaction vessels. Overall, as described in greater detail below, regardless of the multiplex nature of some embodiments of the invention, after amplification of the markers, the method may include processing and sequencing the resulting amplicons to provide information related to the identification, characterization, and strain identity of one or more microorganisms and/or viruses that may be present within the sample.

Some embodiments of the invention may comprise the use of one or more methods of amplifying a nucleic acid-based starting material (i.e., a template, including genomic DNA, crude DNA extract, single-stranded DNA, double-stranded DNA, cDNA, RNA, or any other single-stranded or double-stranded nucleic acids). Nucleic acids may be selectively and specifically amplified from a template nucleic acid contained in a sample. In some nucleic acid amplification methods, the copies are generated exponentially. Examples of nucleic acid amplification methods known in the art include: polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), amplification with Qβ replicase, whole genome amplification with enzymes such as φ29, whole genome PCR, in vitro transcription with T7 RNA polymerase or any other RNA polymerase, or any other method by which copies of a desired sequence are generated.

In addition to genomic DNA, any polynucleotide sequence can be amplified with an appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

PCR generally involves the mixing of a nucleic acid sample, two or more primers or oligonucleotides (primers and oligonucleotides are used interchangeably herein) that are designed to recognize the template DNA, a DNA polymerase, which may be a thermostable DNA polymerase such as Taq or Pfu, and deoxyribose nucleoside triphosphates (dNTP's). In some embodiments, the DNA polymerase used can comprise a high fidelity Taq polymerase such that the error rate of incorrect incorporation of dNTPs is less than one per 1,000 base pairs. Reverse transcription PCR, quantitative reverse transcription PCR, and quantitative real time reverse transcription PCR are other specific examples of PCR. In general, the reaction mixture is subjected to temperature cycles comprising a denaturation stage (typically 80-100° C.), an annealing stage with a temperature that is selected based on the melting temperature (Tm) of the primers and the degeneracy of the primers, and an extension stage (for example 40-75° C.). In real-time PCR analysis, additional reagents, methods, optical detection systems, and devices known in the art are used that allow a measurement of the magnitude of fluorescence in proportion to concentration of amplified template. In such analyses, incorporation of fluorescent dye into the amplified strands may be detected or measured.

Either primers or primers along with probes allow a quantification of the amount of specific template DNA present in the initial sample. In addition, RNA may be detected by PCR analysis by first creating a DNA template from RNA through a reverse transcriptase enzyme (i.e., the creation of cDNA). The marker expression may be detected by quantitative PCR analysis facilitating genotyping analysis of the samples.

“Amplification” is a special case of nucleic acid replication involving template specificity. Amplification may be a template-specific replication or a non-template-specific replication (i.e., replication may be specific template-dependent or not). Template specificity is here distinguished from fidelity of replication (synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out. The amplification process may result in the production of one or more amplicons.

The term “template” refers to nucleic acid originating from a sample that is analyzed for the presence of one or more markers. In contrast, “background template” or “control” is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified out of the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

In addition to primers and probes, template specificity is also achieved in some amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under the conditions in which they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. Other nucleic acid sequences will not be replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (Chamberlin et al. (1970) Nature (228):227). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (Wu and Wallace (1989) Genomics (4):560). Finally, Taq and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H. A. Erlich (ed.) (1989) PCR Technology, Stockton Press).

The term “amplifiable nucleic acid” refers to nucleic acids that may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” will usually comprise “sample template.” The terms “PCR product,” “PCR fragment,” “amplification product,” and “amplicon” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

In some forms of PCR assays, quantification of a target in an unknown sample is often required. Such quantification may be determined in reference to the quantity of a control sample. The control sample starting material/template may be co-amplified in the same tube in a multiplex assay or may be amplified in a separate tube. Generally, the control sample contains template at a known concentration. The control sample template may be a plasmid construct comprising only one copy of the amplification region to be used as quantification reference. To calculate the quantity of a target in an unknown sample, various mathematical models are established. Calculations are based on the comparison of the distinct cycle determined by various methods, e.g., crossing points (C_(P)) and cycle threshold values (C_(t)) at a constant level of fluorescence; or C_(P) acquisition according to established mathematic algorithm.

Some embodiments of the invention may comprise a multiplex assay. As used herein, the term “multiplex” refers to the production of more than one amplicon, PCR product, PCR fragment, amplification product, etc. in a single reaction vessel. In other words, multiplex is to be construed as the amplification of more than one marker-specific sequences within a PCR reaction or assay within the same PCR assay mixture (e.g., more than one amplicon is produced within a single vessel that contains all of the reagents necessary to perform a PCR reaction). In some embodiments, a step prior to performing the PCR (or RT-PCR, quantitative RT-PCR, etc.) reaction can occur such that sets of primers and/or primers and probes are designed, produced, and optimized within a given set of reaction conditions to ensure proper amplicon production during the performance of the PCR.

The algorithm for C_(t) values in real time-PCR calculates the cycle at which each PCR amplification reaches a significant threshold. The calculated C_(t) value is proportional to the number of marker copies present in the sample, and the C_(t) value is a precise quantitative measurement of the copies of the marker found in any sample. In other words, C_(t) values represent the presence of respective marker that the primer sets are designed to recognize. If the marker is missing in a sample, there should be no amplification in the Real Time-PCR reaction.

Alternatively, the C_(p) value may be utilized. A C_(p) value represents the cycle at which the increase of fluorescence is highest and where the logarithmic phase of a PCR begins. The LIGHTCYCLER® 480 Software calculates the second derivatives of entire amplification curves and determines where this value is at its maximum. By using the second-derivative algorithm, data obtained are more reliable and reproducible, even if fluorescence is relatively low.

The various and non-limiting embodiments of the PCR-based method detecting marker expression level as described herein may comprise one or more probes and/or primers. Generally, the probe or primer contains a sequence complementary to a sequence specific to a region of the nucleic acid of the marker gene. A sequence having less than 60% 70%, 80%, 90%, 95%, 99% or 100% identity to the identified gene sequence may also be used for probe or primer design if it is capable of binding to its complementary sequence of the desired target sequence in marker nucleic acid.

Some embodiments of the invention may include a method of comparing a marker in a sample relative to one or more control samples. A control may be any sample with a previously determined level of expression. A control may comprise material within the sample or material from sources other than the sample. Alternatively, the expression of a marker in a sample may be compared to a control that has a level of expression predetermined to signal or not signal a cellular or physiological characteristic. This level of expression may be derived from a single source of material including the sample itself or from a set of sources.

The sample in this method is preferably a biological sample from a subject. The term “sample” or “biological sample” is used in its broadest sense. Depending upon the embodiment of the invention, for example, a sample may comprise a bodily fluid including whole blood, serum, plasma, urine, saliva, cerebral spinal fluid, semen, vaginal fluid, pulmonary fluid, tears, perspiration, mucus and the like; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print, or any other material isolated in whole or in part from a living subject or organism. Biological samples may also include sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes such as blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, and the like. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues.

In some embodiments, sample or biological sample may include a bodily tissue, fluid, or any other specimen that may be obtained from a living organism that may comprise additional living organisms. By way of example only, in some embodiments, sample or biological sample may include a specimen from a first organism (e.g., a human) that may further comprise an additional organism (e.g., bacteria, including pathogenic or non-pathogenic/commensal bacteria, viruses, parasites, fungi, including pathogenic or non-pathogenic fungi, etc.). In some embodiments of the invention, the additional organism may be separately cultured after isolation of the sample to provide additional starting materials for downstream analyses. In some embodiments, the sample or biological sample may comprise a direct portion of the additional, non-human organism and the host organism (e.g., a biopsy or sputum sample that contains human cells and bacteria).

With respect to use of the sample or biological sample, embodiments of the claimed methodology provide improvements compared to conventional methodologies. Specifically, conventional methodologies of identifying and characterizing microorganisms include the need for morphological identification and culture growth. As such, conventional methodologies may take an extended period of time to identify the microorganism and may then require further time to identify whether the microorganism possesses and certain markers. Some embodiments of the invention can provide a user with information about any microorganisms and/or viruses present in a sample without the need for additional culturing because of the reliance of nucleic acid amplification and sequencing. In other words, direct extraction of nucleic acids coupled with amplification of the desired markers and downstream sequencing can reduce significantly the time required to obtain diagnostic and strain identifying information.

The invention may further comprise the step of sequencing the amplicon. Methods of sequencing include but need not be limited to any form of DNA sequencing including Sanger, next-generation sequencing, pyrosequencing, SOLiD sequencing, massively parallel sequencing, pooled, and barcoded DNA sequencing or any other sequencing method now known or yet to be disclosed.

In Sanger Sequencing, a single-stranded DNA template, a primer, a DNA polymerase, nucleotides and a label such as a radioactive label conjugated with the nucleotide base or a fluorescent label conjugated to the primer, and one chain terminator base comprising a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP, are added to each of four reaction (one reaction for each of the chain terminator bases). The sequence may be determined by electrophoresis of the resulting strands. In dye terminator sequencing, each of the chain termination bases is labeled with a fluorescent label of a different wavelength that allows the sequencing to be performed in a single reaction.

In pyrosequencing, the addition of a base to a single-stranded template to be sequenced by a polymerase results in the release of a pyrophosphate upon nucleotide incorporation. An ATP sulfuryrlase enzyme converts pyrophosphate into ATP that in turn catalyzes the conversion of luciferin to oxyluciferin which results in the generation of visible light that is then detected by a camera or other sensor capable of capturing visible light.

In SOLiD sequencing, the molecule to be sequenced is fragmented and used to prepare a population of clonal magnetic beads (in which each bead is conjugated to a plurality of copies of a single fragment) with an adaptor sequence and alternatively a barcode sequence. The beads are bound to a glass surface. Sequencing is then performed through 2-base encoding.

In massively parallel sequencing, randomly fragmented targeted nucleic acids and/or amplicons are attached to a surface. The fragments/amplicons are extended and bridge amplified to create a flow cell with clusters, each with a plurality of copies of a single fragment sequence. The templates are sequenced by synthesizing the fragments in parallel. Bases are indicated by the release of a fluorescent dye correlating to the addition of the particular base to the fragment.

Nucleic acid sequences may be identified by the IUAPC letter code which is as follows: A—Adenine base; C—Cytosine base; G—guanine base; T or U—thymine or uracil base; I—inosine base. M—A or C; R—A or G; W—A or T; S—C or G; Y—C or T; K—G or T; V—A or C or G; H—A or C or T; D—A or G or T; B—C or G or T; N or X—A or C or G or T. Note that T or U may be used interchangeably depending on whether the nucleic acid is DNA or RNA. A sequence having less than 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to the identifying sequence may still be encompassed by the invention if it is able of binding to its complimentary sequence and/or facilitating nucleic acid amplification of a desired target sequence. In some embodiments, as previously mentioned, the method may include the use of massively parallel sequencing, as detailed in U.S. Pat. Nos. 8,431,348 and 7,754,429, which are hereby incorporated by reference in their entirety.

Some embodiments of the invention comprise multiple steps and/or processes that are carried out to execute the universal tail indexing strategy to prepare amplicons corresponding to desired markers for sequencing. In some embodiments, one or more makers for a given sample or template can be selected, as described above. Some embodiments of the invention can be used in conjunction with an analysis of one or more markers (e.g., genes/alleles) associated with a particular phenotype (e.g., virulence).

After selection of the markers, marker-specific primers/oligonucleotides can be designed for the amplification of the markers to produce the desired amplicons, as detailed above. As is known in the art, a forward and a reverse marker-specific primer can be designed to amplify the marker from a nucleic acid sample. In some embodiments, the forward and reverse primers can be designed to produce an amplicon (e.g., some or all of the sequence of the marker) of a desired length. For example, the length of the amplicon may comprise approximately 50 base pairs (bp), 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 1,000 bp, or any size amplicon greater in size or therebetween.

As previously mentioned, some embodiments of the invention may include a multiplex PCR reaction. For example, marker-specific primers can be designed for multiple markers or multiple regions of the same marker such that multiple amplicons of between about 50 bp and 1,000 bp are being produced within a single PCR reaction vessel. In other words, the forward and reverse primers can be designed to function within a given set of temperature parameters such that more than one amplicon can be successfully amplified from a given template within a single PCR reaction mixture. As such, multiple amplicons can be prepared using the universal tail indexing strategy for sequencing preparation.

In some embodiments, the forward and reverse primers that have been designed for each of the markers can be modified to include a universal tail. For example, the universal tail sequences can be relatively or completely unique sequences of nucleotides that are coupled to the 5′ ends of some or all of the forward and reverse marker-specific primers. In some aspects, the universal tail sequences can be selected such that there is little to no overlap in sequence between portions of the markers that are being amplified and the universal tail sequences. Moreover, the universal tail sequences can comprise a length between ten and twenty nucleotides in length. In some embodiments, the universal tail sequences can be any other length, as desired by the user to meet the needs and requirements of the reaction. As such, the universal tail sequences can exhibit a relatively negligible impact on binding of the forward and reverse marker-specific primers to the template sequence to enable amplification. Moreover, as a result of being included on the 5′ end of the forward and reverse marker-specific primers, the universal tail sequences will form a portion of the resulting amplicons. In addition, in some aspects of the invention, the sequences selected for the universal tail sequences can be at least partially correlated with the chemical composition of the template nucleic acids. For example, in some aspects, the sequences selected for the universal tail sequences can be at least partially correlated with the G-C content of the organism from which the template is isolated.

In some aspects, some or all of the universal tail sequences can be at least partially unique. In some embodiments, each of the 5′ ends of all of the forward marker-specific primers within a given PCR assay mixture can comprise the same or a similar universal tail sequence (e.g., a first universal tail sequence or UT1). Similarly, each of the 5′ ends of all of the reverse marker-specific primers within the same PCR assay mixture can comprise a second universal tail sequence (UT2) that differs from the first universal tail sequence. As such, each respective sample from which a template sequence is used in the multiplex PCR assay will have two unique universal tail sequences. Accordingly, each forward and reverse marker-specific primer within a multiplex PCR mixture will include a unique universal tail sequence. For example, if the PCR includes 35 different samples, 35 universal tail sequences can be employed for the forward primers in each of the 35 unique reactions (i.e., not including technical replicates) and 35 universal tail sequences can be employed for the reverse primers in each of the 35 unique reactions (i.e., not including technical replicates). Overall, the forward and reverse marker-specific primers that each comprise the universal tail sequences can comprise a generally short length (e.g., 25-50 bp), which can facilitate simultaneous amplification of multiple targets in a single reaction.

In addition, some embodiments of the invention may comprise performing quantitative PCR to optimize the multiplex PCR assay. For example, after design of the forward and reverse marker-specific primers that each include a universal tail sequence, the contemplated multiplex PCR assays can be performed using quantitative PCR (e.g., using DNA as a template) to assess relative quantities of the amplicons produced. Accordingly, the sequence coverage of each amplicon is considered to be equal if the quantities of the amplicons produced by the multiplex quantitative PCR appear to be equal. If the quantities of the amplicons produced by the multiplex quantitative PCR do not appear to be equal, the forward and/or reverse marker-specific primers can be altered and re-optimized until adequate quantities of amplicons are produced.

After design and adequate optimization of the multiplex PCR assay comprising multiple forward and reverse marker-specific primers that each includes universal tail sequences, the multiplex PCR can be performed to obtain the amplicons associated with the above-described markers. In some embodiments, template that has been previously isolated from a sample can be used for the amplification of the amplicons. In some aspects, multiple PCR reaction replicates can be performed for each sample template and one or more control templates.

In some embodiments, after successful production of the amplicons during the multiplex PCR assay, the resulting amplicons can be further processed to provide sequencing-ready amplicons. For example, some embodiments of the invention may comprise an indexing extension step. In some aspects, the indexing extension step may comprise extending the optimized multiplex amplicons using a set of indexing and common primers that recognize the respective universal tail sequences used for the particular group of amplicons in a minimal cycle PCR assay (e.g., 5-10 total cycles). In particular, each multiplex set of amplicons to be sequenced can be extended with a different set of index oligonucleotides and common oligonucleotides that recognize UT1 and UT2, respectively. In some aspects, the index sequence of the index oligonucleotides can be custom designed to allow for the selection of an index sequence from potentially thousands of different index sequences.

After this step, the resulting products include a set of amplicons for each sample/template that comprise the same index and any necessary sequences that may be required for a particular sequencing platform (e.g., platform sequences associated with the ILLUMINA® Next Generation sequencing platform). Thereafter, the resulting extension-reaction products can be quantified, pooled, and sequenced using a desired platform. In some aspects, the inclusion of the universal tail sequences on the index and common primers can coincide with the use of genomic and index read primers in the mixture of sequencing primer reagents. For example, some embodiments of the invention are capable of pooling multiple amplicons with multiple indices in a single sequencing run to provide 40,000×-95,000× coverage across the amplicons. In other embodiments, the systems and methods associated with the invention can be configured to provide any level of sequencing coverage that is desirable to the user (e.g., higher or lower that the coverage levels discussed above). In some embodiments, after sequencing and generation of the sequence data, the resulting data can be demultiplexed and the sequence files can be aligned to the appropriate references sequences for subsequent sequence analyses.

Embodiments of the invention offer additional advantages relative to conventional systems. For example, some embodiments of the invention comprise the use of PCR before sequencing such that only limited amounts of starting material are necessary and the starting material need not be of high quality (e.g., genomic DNA, crude DNA extracts, single stranded DNA, RNA, cDNA, etc.). In contrast, many conventional sample preparation systems may require relatively large amounts of starting material of relatively high quality, which can limit the use of these systems. Moreover, the inclusion of non-desirable template materials can also interfere in one or more downstream processes in conventional systems and methods. For example, if an investigation is being conducted that focuses on one or more organisms that may be associated with another organism (e.g., bacteria associated with a human); the sampling of the target organism may result in template contamination from the host organism.

In particular, in some aspects, obtaining samples of pathogenic or commensal bacteria from, on, or within a human may also result in the collection of human tissue. As such, when isolating the template, human nucleic acids may contaminate the bacterial template. Some embodiments of the invention are configured such that the contaminating template (e.g., from a human) would not interfere with downstream processes, including sequencing. For example, some embodiments of the invention operate such that only a limited amount of starting template (e.g., 500 femtograms or greater) can be used. Moreover, some embodiments are also configured such that the starting material (e.g., template contaminated with foreign nucleic acids) can still produce the required amplicons for sequencing in the presence of more than a 1,000-fold excess of contaminating template with no discernible inhibition of the multiplex PCR.

In certain aspects, the present invention provides an assay that works with as little as about 1 pg, about 900 fg, about 800 fg, about 700 fg, about 600 fg, about 500 fg, about 400 fg, about 300 fg, about 200 fg, or about 100 fg of genomic DNA.

The following examples are given for purely illustrative and non-limiting purposes of the present invention.

EXAMPLES Example 1. Multiplex Assays for LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay

In one aspect, the LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay involves the steps of DNA or RNA extraction, amplification and library preparation, next-generation sequencing (NGS sequencing), reference mapping, and clinical interpretation as shown in FIG. 1. Amplification and library preparation can be efficiently carried out with multiplex assays of various configurations.

In one aspect, the LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay comprises the configuration of multiplex assays with the following primers identified in Table 1 without universal tails and in Table 2 and Table 3 with universal tails.

-   -   Multiplex 1 assays: 16S-1_UT, 16S-2_UT, 16S-3_UT, 16S-4_UT.         16S-5_UT, Ana-msp2_UT1, bbk32_UT, dbpA_UT, dbpB_UT, Ehrl-16S_UT,         Ehrl-sodB_UT, EV-D68_UT, flaB_UT1, flaB_UT2, Ft-G_UT, glpQ_UT1,         IGS-5S-23S-TK_UT, IGS1-Bunikis_UT1, IGS2-Derdakova_UT,         ospA-Rudenko_UT, ospB_UT1, ospB_UT2, ospC-Bunikis_UT1, ospD_UT1,         p66_UT1, parA_UT1, Yp3a_UT, Yppla_UT, H3N2_UT, Bart-ssrA_UT1,         Babe-18S_UT1, IPC-gapDH_UT1, and Sa_M4_UT1.         When this configuration of the multiplex assays is used, an         amplification reaction mixture is prepared. After the         amplification reactions are complete next-generation sequencing         is carried out to determine the sequences of the amplicons. The         sequences may be analyzed with reference mapping and further         analyzed to arrive at a clinical interpretation.

As shown in FIG. 2, the configuration of Multiplex 1 assays includes:

-   -   1) Borrelia genus-wide assays targeting the Borrelia 16S rRNA,         the 16S-23S intergenic spacer (IGS), and the flaB gene (flagella         subunit B);     -   2) Borrelia burgdorferi sensu lato-specific assays targeting the         adhesin genes (e.g., bbk32, dbpA, and dbpB) outer surface         protein genes (e.g. ospA, ospB, and ospC), and p66 porin genes;     -   3) a non-Lyme Borrelia spp. assay targeting the glpQ gene;     -   4) assays specific to other tick-borne pathogens including         Erlichia spp., Anaplasma phagocytophilum, Babesia spp.,         Bartonella spp., Powassan and deer tick viruses, and Rickettsia         spp.;     -   5) Lyme-like differential diagnostic assays specific to         Staphylococcus aureus, Yersinia pestis, Influenza virus,         Enterovirus, and Francisella tularensis; and     -   6) an internal control assay targeting the human GAPDH gene.

The amplification and sequencing of regions of the flaB gene allows for the differentiation of the tick-borne relapsing fever (TBRF) species group from the Borrelia burgdorferi sensu lato group as shown in FIG. 3. The primers of the multiplex assays are designed to detect all Borrelia species and are located in conserved regions. Comparison of the sequenced amplicons from a sample are compared to an alignment of 482 known unique flaB gene DNA sequences to determine the presence or absence of particular Borrelia species that contribute to disease states such as Lyme disease and relapsing fever (see FIG. 4). Similarly, sequencing of amplicons from five assays covering the majority of the gene sequence of the Borrelia 16S rDNA and comparison of the sequences detected in a sample to an alignment of 185 known unique DNA sequences facilitates detection of Borrelia species in the TBRF species group and in the Borrelia burgdorferi sensu lato (Lyme) group (see FIG. 5 and FIG. 6).

Example 2. Sensitivity Results with LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay

Eight strains of Borrelia burgdorferi sensu lato were serially diluted and the DNA extracted from each diluted strain. After amplification using primers from Table 3 and next-generation sequencing the results showed that each strain was properly identified and the number of sequence reads mapping to the 16S rRNA reference correlated with the dilution factor of each sample.

Example 3. Detection of Borrelia Species in DNA Extracted from Ixodes pacificus

Seventy-four Western black-legged tick (Ixodes pacificus) samples were collected form the San Francisco Bay area and the DNA of each sample was extracted and analyzed as described in Example 1 with the following primers from Table 2:

-   -   16S_UT, IGS2-5S-23S-TK_UT, IGS1-Bunikis_UT1, bbk32_UT, dbpA_UT,         dbpB_UT, flaB_UT1, flaB_UT2, glpQ_UT1, ospA-Rudenko_UT,         ospB_UT2, ospC-Bunikis_UT1, ospD_UT1, p66_UT1, parA_UT1,         IPC-gapDH_UT1, Ana-msp2_UT1, Ehrl-16S_UT, Ehrl-sodB_UT,         EV-D68_UT, Ft-G_UT, Yp3a_UT, Yppla_UT, H3N2_UT, Bart-ssrA_UT1,         Babe-18S_UT1, Sa_M4_UT1         After the amplicons were sequenced and analyzed with the LymeSeq         Lyme Disease Next-Generation Sequencing Diagnostic Assay         twenty-seven samples were found to contain genomic DNA from         Borrelia burgdorferi sensu lato, eight samples contains genomic         DNA from B. miyamotoi, one sample contained genomic DNA from         Bartonella spp., and one sample contained genomic DNA from         Anaplasma phagocytophilum.

Example 4. Investigation of Tick-Borne Relapsing Fever Outbreak with LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay

Borrelia hermsii is one of the species causing tick-borne relapsing fever (TBRF) in infected patients. An outbreak of TBRF was investigated in Northern Arizona (see Jones, J M et al., “Tick-Borne Relapsing Fever Outbreak among a High School Football Team at an Outdoor Education Camping trip, Arizona, 2014,” Am. J. Trop. Med. Hyg. 95(3), 2016, pp. 546-550). Blood was collected from several patients who were febrile after recent tick exposure. Eight blood samples were analyzed with the LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay as described in Example 1, and the assay indicated that seven of the eight were positive for Borrelia hermsii. TBRF was confirmed in several of these patients by spirochetemia detection on blood smear and/or by culturing blood samples from the patients and isolating Borrelia hermsii.

Example 5. Analysis of DNA Versus RNA with LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay in Spiked Blood Samples and TBRF Outbreak Blood Samples

Blood samples were spiked with Borrelia burgdorferi and subsequently analyzed with the LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay. Amplicon sequencing allows for analysis of extracted RNA as well as DNA. Both DNA and RNA were extracted from the spiked blood samples and analyzed as described in Example 1. The colony forming units (CFU) of Borrelia burgdorferi were counted in each spiked blood sample and plotted against the number of sequence reads for 16S rRNA, flaB-1, flaB-2, and ospB from each sample of extracted RNA or DNA (see FIG. 7). The results showed evidence of 16S rRNA in blood at a relatively high level even when very few Borrelia burgdorferi CFUs were present suggesting that extraction and analysis of RNA samples increased the sensitivity of the LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay as compared to extraction and analysis of DNA samples.

In another experiment, eight blood samples known to contain Borrelia hermsii were analyzed with the LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay. DNA and RNA from each sample were analyzed. The assay confirmed the presence of Borrelia hermsii in all eight samples. In addition, the sequence reads from the extracted RNA samples were generally greater than those from the corresponding extracted DNA samples. For instance, in one example the extracted DNA produced only 200 sequence reads while the corresponding extracted RNA produced 200,000 sequence reads. These results confirmed the enhanced sensitivity of the assay when used to analyze RNA samples.

Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes.

It is understood that the disclosed invention is not limited to the particular methodology, protocols and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

TABLE 1 Universal tail targets and assays for LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay. The primers listed do not include the universal tails (UT). Target SEQ Target Target Gene/ Sequence without ID Purpose Taxon Region UT Assay Type Assay Primer UT NO: Species Borrelia 16S- Sequence-based 16S-1_UT 16S-1_UT_F CGGGTGAGTAAC 1 ID spp. set of GCGTGGAT 5 assays to cover whole gene Also good RNA 16S-1_UT_R CCTCTCAGGCCG 2 target GTTACTTATC 16S-2_UT 16S-2_UT_F CGGTCACACTGG 3 AACTGAGA 16S-2_UT_R GCTGCTGGCACG 4 TAATTAGC 16S-3_UT 16S-3_UT_F GCGAGCGTTGTT 5 CGGGAT 16S-3_UT_R ACTCAGCGTCAG 6 TCTTGACC 16S-4_UT 16S-4_UT_F CGCTGTAAACGA 7 TGCACACTTG I6S-4_UT_R ACAACCATGCAG 8 CACCTGTA 16S-5_UT 16S-5_UT_F GCAACGAGCGCA 9 ACCCTT 16S-5_UT_R TACAAGGCCCGA 10 GAACGTATTCAC Species Borrelia IGS2 5S- Sequence-based IGS2-5S-23S- IGS-5S-23S- GAGTTCGCGGGA 11 ID spp. 23S TK_UT TK_UT_F GAGTAGGTTATT GCC rrfA-rrlB Also good RNA IGS-5S-23S- TCAGGGTACTTA 12 target TK_UT_R GATGKTTCACTT CC IGS2-5S-23S- IGS-5S-23S- CTGCGAGTTCGC 13 Postic_UT Postic_UT_F GGGAGA IGS-5S-23S- TCCTAGGCATTC 14 Postic_UT_R ACCATA IGS2- IGS2- CGACCTTCTTCGC 15 Derdakova_UT Derdakova_UT CTTAAAGC F IGS2- AGCTCTTATTCGC 16 Derdakova_UT TGATGGTA R Species Borrelia IGS1 Sequence-based IGS1- IGS1- GTATGTTTAGTG 17 ID spp. Bunikis_UT1 Bunikis_UT1_F AGGGGGGTG rrs-rrlA Also good RNA IGS1- GGATCATAGCTC 18 target Bunikis_UT1 AGGTGGTTAG R IGS1- IGS1- AGGGGGGTGAAG 19 Bunikis_UT2 Bunikis_UT2_F TCGTAACAAG IGS1- GTCTGATAAACC 20 Bunikis_UT2 TGAGGTCGGA R Species Borrelia IGS rrs-rrlA rrs-rrlA_UT1 rrs- GGGTTCGAGTCC 219 ID spp. 16S-23S rrlA_UT1_F1 CTYAACCT IGS rrs- TTGGTTTAGAGC 220 rrlA_UT1_F2 ATCGGCTTTGC rrs- CCTTGCACTTTAG 221 rrlA_UT1_R1 CGAAACAAC rrs- CCTTGTGCTTTAG 222 rrlA_UT1_R2 TGAAACAAC rrs- ACTTGCCATACG 223 rrlA_UT1_R3 TAAACAACCGT rrs- CTCATGACTTGTC 224 rrlA_UT1_R4 ACACGTAAACAA C rrs- GTTCAACTCCTCC 225 rrlA_UT1_R5 TGGTCCCAA rrs- ATCCTATAGATG 226 rrlA_UT1_R6 CAATCTCTTGWC C rrs- TTTGCATGTAATC 227 rrlA_UTI_R7 AAGTCTTGGAAT TC rrs- TACTTTCACCTCT 228 rrlA_UTI_R8 AGACATTCTTGT rrs- TAGGTTGATTCA 229 rrlA_UTI_R9 TGATCAGGTCCT T rrs- CGATTCGGTCAC 230 rrlA_UTI_R1O GGCTCTTAC rrs- CCTTATGATTTAG 231 rrlA_UTI_R11 TAACACAACGTA AGT rrs- AAGCTAGTAATG 232 rrlA_UTI_R12 AATGTGGGATGT T Species Borrelia flaB Sequence-based flaB_UT1 flaB_UTI_F GCWTCTGATGAT 21 ID spp. GCTGCTGGIA flaB_UTI_R1 GCATTCCAAGYT 22 CTTCAGCTGT flaB_UTI_R2 GCATTCCAAGCT 23 CTTCAGCWGT flaB_UT2 flaB_UT2_F1 ACACCAGCRTCR 24 CTTTCAGG flaB_UT2_F2 ACACCAGCATCA 25 YTAKCTGGA flaB_UT2_F3 ACACCAGCATCA 26 TTRGCTGGA flaB_UT2_R1 TTGGAAAGCACC 27 TAAATTTGCYCTT flaB_UT2_R2 TTGRAAAGCACC 28 AAGATTTGCTCTT flaB_UT2_R3 TTGGAAAGCACC 29 YAAATTTGCTCTT Species non- glpQ Sequence-based glpQ_UT1 glpQ_UTI_F1 CCAGAACATACC 30 ID Burgdorferi TTAGAAKCTAAA Borrelia GC spp glpQ_UTI_F2 CAGAACATACAT 31 TAGAAGCCAAAG C glpQ_UTI_R1 CCTTGTTGYTTAT 32 GCCATAAKGGTT glpQ_UTI_R2 CCTTGTTGTTTAT 33 GCCAHAAGGGTT glpQ- glpQ- CCAGAACATACC 34 Halp_UT2 Halp_UT2_F1 TTAGAAKCTAAA GC glpQ- CAGAACATACAT 35 Halp_UT2_F2 TAGAAGCCAAAG C glpQ- CACATTAGCAGA 36 Halp_UT2_R1 AATCAAATCAC glpQ- GATCAAATCTTT 37 Halp_UT2_R2 CGCTAAGRCTTA GTG glpQ- GATCAAATCTTT 38 Halp_UT2_R3 CACTGAGACTTA GTG glpQ- GATCAAATCTTT 39 Halp_UT2_R4 CACTAAGGCTTA ATG glpQ- GGGTATCCARGG 40 Halp_UT2_R5 TCCAAT Species B. bbk32 presence/absence bbk32_UT bbk32_UT_F1 TGGAGGAGMCTA 41 ID burgdorferi TTGAAAGYAATG sensu stricto Also good RNA bbk32_UT_F2 TGAAGGAKACTA 42 target TTGAAAGYAATG bbk32_UT_R1 GCGTGTAGAATA 43 CATTTGGGTTAG C bbk32_UT_R2 GACGTGTAGAAT 44 ACATTTGGGTTT GC Species B. dbpA presence/absence dbpA_UT dbpA_UT_F AACAATGTAAAT 45 ID burgdorferi TTTGCTGCCTTT Also good RNA dbpA_UT_R CCTGAGACCTCA 46 target AGCATCAT Species B. dbpB presence/absence dbpB_UT dbpB_UT_F CGGTTCCAAGGT 47 ID burgdorferi AACAAGTG Also good RNA dbpB_UT_R TAATCCAATACT 48 target ACATGCGACCAA TA Species B. dbpA presence/absence dbpA_UT2 dbpA_UT2_F1 CAGCCGCATCTG 233 ID burgdorferi TAACTG dbpA_UT2_F2 TCAGTTCCCATTG 234 AAACTG dbpA_UT2_F3 TTYAGCYGCATC 235 TGAGAC dbpA_UT2_F4 TTCAGCTGCCWT 236 TGAGAC dbpA_UT2_R1 CAGGYAGCAAGG 237 TATCAGA dbpA_UT2_R2 CRGGTAGYGGGG 238 TATCAGA dbpA_UT2_R3 AACAGGTRGAAA 239 GGYAGCA Species B. dbpB presence/absence dbpB_UT2 dbpB_UT2_F1 CGCAAGCAATCT 240 ID burgdorferi TTCAGYTGTGT dbpB_UT2_F2 CTCAACCAATCT 241 TTCAGCYGTGT dbpB_UT2_F3 CTTCAAGCAATC 242 TTTCACATGTGT dbpB_UT2_F4 CCTCAATTAATCT 243 TTCAGATGTGCT dbpB_UT2_F5 TTCAAGCAATCT 244 TTCGGCTGTGT dbpB_UT2_F6 CTCCATTACTCTT 245 TCGGCTGTGT dbpB_UT2_R1 RYAGCKCTTGAA 246 TCRTCYTYTAAG G dbpB_UT2_R2 AAGCAATGCTTG 247 AATCSTMTTCTG A dbpB_UT2_R3 AAGCAAAGCTTG 248 AATCGTCTTCC Species Anaplasma msp2 presence/absence Ana- Ana- AGTTTGACTGGA 49 ID phagocyto- (major msp2_UT1 msp2_UT1_F ACACWCCTGATC philum surface protein) AY151054 Ana- CTCGTAACCAAT 50 msp2_UT1_R CTCAAGCTCAAC Species Anaplasma msp2 presence/absence Ana- Ana- GGGAGAGTAACG 51 ID phagocyto- (major msp2_UT2 msp2_UT2_F GAGARACWAAG philum surface G protein) Ana- CTGGCACCACCA 52 msp2_UT2_R1 ATACCATAACC Ana- CTGGCACCACCA 53 msp2_UT2_R2 ATACCRTACC Ana- GGGAGAGTAACG 54 msp2_UT2_F GAGARACWAAG G Ana- CTCGTAACCAAT 55 msp2_UT1_R CTCAAGCTCAAC Species Ehrlichia 16S presence indicates Ehrl-_16S_UT EhrI-16S_ GAGGATTTTATC 56 ID genus genus present UT_F TTTGTATTGTAGC TAAC sequence tells Ehrl- TGTAAGGTCCAG 57 species 16S_UT_R CCGAACTGACT Species Ehrlichia sodB presence/absence Ehrl-sodB_UT Ehrl- TTTAATAATGCT 58 ID genus sodB_UT_F GGTCAAGTATGG AATCAT sequence-based to Ehrl- AAGCRTGYTCCC 59 tell species sodB_UT_R ATACATCCATAG Species B. ospB presence/absence ospB_UT_1 ospB_UT_F1 TGCGGTGACAGA 60 ID burgdorferi AGACTC ospB_UT_R1 CAGCAGAAACTG 61 TTAATTTTACTTT ACTC presence/absence ospB_UT_2 ospB_UT_F2 TGCGGTGACAGA 62 AGACTC ospB_UT_R2 AATCAGCAGAAA 63 CTGTTAATTTTAC TTTAC Species B. ospB presence/absence ospB_UT3 ospB_UT3_F1 GTYGAACTTAAA 249 ID burgdorferi GGAACTTCCGAT ospB_UT3_F2 NTTGAGCTWAAA 250 GGAACWTCTGAT ospB_UT3_F3 GTTGAGCTTAAA 251 GGRGTTKCTGA ospB_UT3_F4 GGTGAGCTTAAA 252 GGGGATTTTGA ospB_UT3_F5 GTTGAGCTTAAA 253 GGCCTTTCTGAG ospB_UT3_R1 CCGMCTMCAAG 254 ACTTCCTTCA ospB_UT3_R2 CCGCCTACAAGA 255 TTTCCTGGA ospB_UT3_R3 CCACCAACAAGA 256 CTTCCTTCTAGT ospB_UT3_R4 CCACCAACTAGA 257 CTTCCTTTAAAC ospB_UT3_R5 CCACCAACAAGA 258 TTTCCTTCGAAC ospB_UT3_R6 CATTAGCTACTTT 259 TCCTTCAAGAG ospB_UT3_R7 CATTAGCTAGAG 260 TTCCTTCAAGAG ospB_UT3_R8 TCAGCAGYTAGA 261 GTTCCTTCAAGA Species B. ospC-TG presence/absence ospC-TG_UT1 ospC- TCAGGRAAAGAT 262 ID burgdorferi TG_UT1_F GGGAATRCATCT GC ospC- GRCTTGTAAGCT 263 TG_UT1_R CTTTAACTGMAT TAG Species B. p66 presence/absence p66_UT3 p66_UT3_F1 GCCYATGACYGG 264 ID burgdorferi ATTCAAA p66_UT3_F2 TTYGCACCTATG 265 ACTGGRTTT p66_UT3_R GGYTTCCATGTT 266 GCTTGAAY p66_UT4 p66_UT4_F1 TGARGCTATCCA 267 TCCAAGRCC p66_UT4_F2 GAAGCTGTCCAT 268 CCAAGATTAG p66_UT4_R1 CGGTTTAGCTTG 269 GAATACAGATGA p66_UT4_R2 CGGTTTTGCCTG 270 GAATAAAGATGA p66_UT4_R3 GGCYTAGCTTGG 271 AAYATAGATGA p66_UT5 p66_UT5_F GCAATMGGAAA 272 YTCAACATTC p66_UT5_R CRCTTGCAAATG 273 GGTCTATTCCT Species B. ospA ospA ospA_UT1 ospA_UT1_F1 GGITCTGGAAYA 274 ID burgdorferi CTTGAAGG ospA_UT1_F2 GGATCTGGRRTR 275 CTTGAAGG ospA_UT1_F3 GGTTCTGGAASC 276 CTTGARGG ospA_UT1_F4 GGRYCTGGGGTR 277 CTTGAAGG ospA_UT1_F5 GGATCTGGGGGA 278 AAGCTTGAAG ospA_UT1_F6 GGTTCTGGDGTR 279 CTKGAAGG ospA_UT1_F7 GGATCTGGMWH 280 GCYYGAAGG ospA_UT1_F8 GGMGCTGGAMA 281 WCTTGAAGG ospA_UT1_R1 CAAGTYTGKTKC 282 CRTTTKCTCTTG ospA_UT1_R2 CAAGYYTGGTWC 283 CGTYTGCTCTTR ospA_UT1_R3 CMAGTGTAGTYC 284 CGYTTGDTCTTG ospA_UT1_R4 CAAGTMTKGWW 285 CCRTTTGCTCTTR ospA_UT1_R5 CAAGKGTAGTTT 286 CGTTTKCTCTTG ospA_UT1_R6 CAAKTGTAGTAT 287 YRTTTGATCTTG ospA_UT1_R7 CAAGMKTRGTKC 288 CGTTTGCTCTTG ospA_UT1_R8 CAAGTCTGGTTC 289 CGTCTTTTCTTG ospA_UT1_R9 CAAGTGGTGTTC 290 CGTTTGTTCTTG ospA_UT1_R10 CAAGTCTATTTCC 291 ATTTGCTCTTG ospA_UT1_R11 CAAGTCTGGTTC 292 CGTTAYCTCTTA ospA_UT1_R12 CAAGTCTGGTTC 293 CATTTGCCCTTA Species B. ospC presence/absence ospC- ospC- ATGAAAAAGAAT 64 ID burgdorferi Bunikis_UT1 Bunikis_UT1_F ACATTAAGTGC Also and sequence-based ospC- ATTAATCTTATA 65 typing Bunikis_UT1 ATATTGATTTTAA info R TTAAGG presence/absence ospC- ospC- TATTAATGACTTT 66 Bunikis_UT2 Bunikis_UT2_F ATTTTTATTTATA TCT and sequence-based ospC- TTGATTTTAATTA 67 Bunikis_UT2 AGGTTTTTTTGG R presence/absence ospC- ospC- AAAGAATACATT 68 Wang_UT1 Wang_UT1_F AAGTGCGATATT and sequence-based ospC- GGGCTTGTAAGC 69 VVang_UT1_R TCTTTAACT Species B. p66 presence/absence p66- p66- GATTTTTCTATAT 70 ID burgdorferi Bunikis_UT1 Bunikis_UT1_F TTGGACACAT Also good RNA p66- TGTAAATCTTATT 71 target Bunikis_UT1_ AGTTTTTCAAG R presence/absence p66- p66- CAAAAAAGAAAC 72 Bunikis_UT2 Bunikis_UT2_F ACCCTCAGATCC Also good RNA p66- CCTGTTTTTAAAT 73 target Bunikis_UT2_R AAATTTTTGTAG CATC presence/absence p66- p66- CGAAGATACTAA 74 Rudenko_UT1 Rudenko_UT1_F ATCTGT Also good RNA p66- GCTGCTTTTGAG 75 target Rudenko_UT1_R ATGTGTCC Species B. ospA presence/absence ospA- ospA- GAGCTTAAAGGA 76 ID burgdorferi Rudenko_UT Rudenko_UT_F ACTTCTGATAA ospA- GTATTGTTGTACT 77 Rudenko_UT_R GTAATTGT Differen- Enterovirus VP1 presence/absence EV-D68_UT EV- ACCAGARGAAGC 78 tial strain D68 D68_UT_F1 CATACAAAC diagnos- tic EV- TGACACTTCAAG 79 D68_UT_F2 CAATGTTCGTA EV- AACGCCGAACTT 80 D68_UT_F3 GGTGTG EV- AACACCGAACCA 81 D68_UT_F4 GAGGAAG EV- SCTGAYTGCCAR 82 D68_UT_R1 TGGAATGAA EV- ATGTGCTGTTATT 83 D68_UT_R2 GCTACCTACTG EV- ATTATTACTACTA 84 D68_UT_R3 CCATTCACTGCT ACA EV- TCAAATCCAGCA 85 D68_UT_R4 AAGCCATCA EV- AGAATACACTAG 86 D68_UT_R5 CATTACTACCTG ACT Differen- Staphylo- Sa_M4_UT2 Sa_M4_UT2_F TAGCGTTGGTAT 87 tial coccus TAAGTGGTTGT diagnos- aureus tics Sa_M4_UT2_R GTCATAGCATAG 88 TTCGGGTCA Differen- Influenza matrix gene presence/absence H3N2_UT H3N2_UT_F AAGACCAATYCT 89 tial GTCACCTCTGA diagnos- tics RNA target H3N2_UT_R CAAAGCGTCTAC 90 GCTGCAGTCC Differen- Yersinia plasmid Yppla_UT Yppla_UT_F GAAAGGAGTGCG 91 tial pestis GGTAATAGGTT diagnos- tics Yppla_UT_R GGCCTGCAAGTC 92 CAATATATGG chromosome Yp3a_UT Yp3a_UT_F CATTGGACGGCA 93 TCACGAT Yp3a_UT_R AGTTGGCCAGCG 94 ATTCGA Differen- Francisella SNP Ft-G_UT Ft-G_UT_F CTAAGCCATAAG 95 tial tularensis CCCTTTCTCTAAC diagnos- TTGT tics Ft-G_UT_R AGCAATGACAAA 96 GCTTGTTGAAAA AG Species Borrelia porin gene presence/absence p66- p66_UT1_F GTAATTGCAGAA 97 ID burgdorferi borrelia_UT1 ACACCTTTTGAA T p66_UT1_R CTGCTTTTGAGAT 98 GTGTCCAA presence/absence p66- p66_UT2_F TGTAATTGCAGA 99 borrelia_UT2 AACACCTTTTGA p66_UT2_R gctgcttttgag 100 ATGTGTCC Species outer presence/absence ospD- ospD_UT1_F ATCAWMTGAGG 101 ID surface borrelia_UT1 CAAATAAAGTTG protein D TAGA ospD_UT1_R TGTTCTGCYGCTT 102 TAGTAAGG Species Borrelia partitioning presence/absence par_A_UT1 par_A_UT1_F TTRACTTCTTCTA 103 ID burgdorferi gene TYGCATCCATTA par_A_UT1_R TRTTCCTTCTCAT 104 CCAATTCTATGT Genus ID Bartonella ssrA presence/absence Bart-ssrA_UT1 Bart- GGCTAAATIAGTA 105 ssrA_UT1_F GTTGCAAAYGAC A Bart- GCTTCTGTTGCCA 106 ssrA_UT1_R GGTG Genus ID Babesia 18S sequence-based Babe-18S_UT1 Babe- ACCGTCCAAAGC 107 18S_UT1_F TGATAGGTC Babe- CGAAACTGCGAA 108 18S_UT1_R TGGCTCATTA Genus ID Rickettsia ompA presence/absence Rkttsia- Rkttsia- GGCATTTACTTA 294 ompA_UT1 ompA_UT1_F CRGTGSTGAT Rkttsia- CCATGATTTGCA 295 ompA_UT1_R GCAAYAGCAT Rkttsia- Rkttsia- CGYTAGCTGGGC 296 ompA_UT2 ompA_UT2_F TTAGRTATTC Rkttsia- CGCCGRAACTTT 297 ompA_UT2_R ATTCTTGAATG Rkttsia- Rkttsia- ACTTAYGGTGGT 298 ompA_UT3 ompA_UT3_F GATTATAYTATC Rkttsia- TGCAGCAACAGC 299 ompA_UT3_R ATTAKTACYG Rkttsia- Rkttsia- GCTGRAGGAGTA 300 ompA_UT4 ompA_UT4_F1 GCTAATGGT Rkttsia- GCAGCAGGAGTA 301 ompA_UT4_F2 GCTGATGAT Rkttsia- MCGCAGCAGTAC 302 ompA_UT4_R CGGTTAAAG Rkttsia- Rkttsia- CAACCGCAGCRW 303 ompA_UT5 ompA_UT5_F TAATGCTAAC Rkttsia- CCTCCCGTATCTA 304 ompA_UT5_R CCACTGAAC Rkttsia- Rkttsia- TGCAGGAGCAGA 305 ompA_UT6 ompA_UT6_F TAATGGTA Rkttsia- GCCGGCAGTAAT 306 ompA_UT6_R AGTAACAG Rkttsia- Rkttsia- GGTGCAAGCCAA 307 ompA_UT7 ompA_UT7_F1 GTAACATATAC Rkttsia- AGGTACAAATCA 308 ompA_UT7_F2 AGTAACATATAC C Rkttsia- AAACCGCCTTCC 309 ompA_UT7_R1 GTTTCTG Rkttsia- AATCCACCTGCC 310 ompA_UT7_R2 GCTTCTG Genus ID Powassan presence/absence Powass_UT Powass_UT_F1 GGCDGTAGGYCA 311 and deer TGTTTATGAC tick viruses Powass_UT_F2 AGCTGTGGGCCA 312 CGTCTATGAC Powass_UT_R1 CCGAAGGCAGGT 313 GATCTTTG Powass_UT_R2 CAGAAGGCAGGT 314 GGTCCTTG Internal Human gapDH presence/absence IPC- IPC- CCTGCCAAATAT 109 control gapDH_UT1 gapDH_UT1_F GATGACATCAAG IPC- GTGGTCGTTGAG 110 gapDH_UT1_R GGCAATG

TABLE 2 Primers for LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay with the universal tail targets. ACCCAACTGAATGGAGC (SEQ ID NO: 217) or ACGCACTTGACTTGTCTTC (SEQ ID NO: 218). Sequence with SEQ Universal Tail ID Primer Target NO: 16S-1_UT_F ACCCAACTGA 111 ATGGAGCCGG GTGAGTAACG CGTGGAT 16S-1_UT_R ACGCACTTGA 112 CTTGTCTTCC CTCTCAGGCC GGTTACTTAT C 16S-2_UT_F ACCCAACTGA 113 ATGGAGCCGG TCACACTGGA ACTGAGA 16S-2_UT_R ACGCACTTGA 114 CTTGTCTTCG CTGCTGGCAC GTAATTAGC 16S-3_UT_F ACCCAACTGA 115 ATGGAGCGCG AGCGTTGTTC GGGAT 16S-3_UT_R ACGCACTTGA 116 CTTGTCTTCA CTCAGCGTCA GTCTTGACC 16S-4_UT_F ACCCAACTGA 117 ATGGAGCCGC TGTAAACGAT GCACACTTG 16S-4_UT_R ACGCACTTGA 118 CTTGTCTTCA CAACCATGCA GCACCTGTA 16S-5_UT_F ACCCAACTGA 119 ATGGAGCGCA ACGAGCGCAA CCCTT 16S-5_UT_R ACGCACTTGA 120 CTTGTCTTCT ACAAGGCCCG AGAACGTATT CAC Ana-msp2_UT1_F ACCCAACTGA 121 ATGGAGCAGT TTGACTGGAA CACWCCTGAT C Ana-msp2_UT1_R ACGCACTTGA 122 CTTGTCTTCC TCGTAACCAA TCTCAAGCTC AAC Ana-msp2_UT2_F ACCCAACTGA 123 ATGGAGCGGG AGAGTAACGG AGARACWAAG G Ana-msp2_UT2_R1 ACGCACTTGA 124 CTTGTCTTCC TGGCACCACC AATACCATAA CC Ana-msp2_UT2_R2 ACGCACTTGA 125 CTTGTCTTCC TGGCACCACC AATACCRTAC C Babe-18S_UT1_F ACCCAACTGA 126 ATGGAGCACC GTCCAAAGCT GATAGGTC Babe-18S_UT1_R ACGCACTTGA 127 CTTGTCTTCC GAAACTGCGA ATGGCTCATT A Bart-ssrA_UT1_F ACCCAACTGA 128 ATGGAGCGGC TAAATTAGTA GTTGCAAAYG ACA Bart-ssrA_UT1_R ACGCACTTGA 129 CTTGTCTTCG CTTCTGTTGC CAGGTG bbk32_UT_F1 ACCCAACTGA 130 ATGGAGCTGG AGGAGMCTAT TGAAAGYAAT G bbk32_UT_F2 ACCCAACTGA 131 ATGGAGCTGA AGGAKACTAT TGAAAGYAAT G bbk32_UT_R1 ACGCACTTGA 132 CTTGTCTTCG CGTGTAGAAT ACATTTGGGT TAGC bbk32_UT_R2 ACGCACTTGA 133 CTTGTCTTCG ACGTGTAGAA TACATTTGGG TTTGC dbpA_UT_F ACCCAACTGA 134 ATGGAGCAAC AATGTAAATT TTGCTGCCTT T dbpA_UT_R ACGCACTTGA 135 CTTGTCTTCC CTGAGACCTC AAGCATCAT dbpB_UT_F ACCCAACTGA 136 ATGGAGCCGG TTCCAAGGTA ACAAGTG dbpB_UT_R ACGCACTTGA 137 CTTGTCTTCT AATCCAATAC TACATGCGAC CAATA Ehrl-16S_UT_F ACCCAACTGA 138 ATGGAGCGAG GATTTTATCT TTGTATTGTA GCTAAC Ehrl-16S_UT_R ACGCACTTGA 139 CTTGTCTTCT GTAAGGTCCA GCCGAACTGA CT Ehrl-sodB_UT_F ACCCAACTGA 140 ATGGAGCTTT AATAATGCTG GTCAAGTATG GAATCAT Ehrl-sodB_UT_R ACGCACTTGA 141 CTTGTCTTCA AGCRTGYTCC CATACATCCA TAG EV-D68_UT_F1 ACCCAACTGA 142 ATGGAGCACC AGARGAAGCC ATACAAAC EV-D68_UT_F2 ACCCAACTGA 143 ATGGAGCTGA CACTTCAAGC AATGTTCGTA EV-D68_UT_F3 ACCCAACTGA 144 ATGGAGCAAC GCCGAACTTG GTGTG EV-D68_UT_F4 ACCCAACTGA 145 ATGGAGCAAC ACCGAACCAG AGGAAG EV-D68_UT_R1 ACGCACTTGA 146 CTTGTCTTCS CTGAYTGCCA RTGGAATGAA EV-D68_UT_R2 ACGCACTTGA 147 CTTGTCTTCA TGTGCTGTTA TTGCTACCTA CTG EV-D68_UT_R3 ACGCACTTGA 148 CTTGTCTTCA TTATTACTAC TACCATTCAC TGCTACA EV-D68_UT_R4 ACGCACTTGA 149 CTTGTCTTCT CAAATCCAGC AAAGCCATCA EV-D68_UT_R5 ACGCACTTGA 150 CTTGTCTTCA GAATACACTA GCATTACTAC CTGACT flaB_UT1_F ACCCAACTGA 151 ATGGAGCGCW TCTGATGATG CTGCTGGTA flaB_UT1_R1 ACGCACTTGA 152 CTTGTCTTCG CATTCCAAGY TCTTCAGCTG T flaB_UT1_R2 ACGCACTTGA 153 CTTGTCTTCG CATTCCAAGC TCTTCAGCWG T flaB_UT2_F1 ACCCAACTGA 154 ATGGAGCACA CCAGCRTCRC TTTCAGG flaB_UT2_F2 ACCCAACTGA 155 ATGGAGCACA CCAGCATCAY TAKCTGGA flaB_UT2_F3 ACCCAACTGA 156 ATGGAGCACA CCAGCATCAT TRGCTGGA flaB_UT2_R1 ACGCACTTGA 157 CTTGTCTTCT TGGAAAGCAC CTAAATTTGC YCTT flaB_UT2_R2 ACGCACTTGA 158 CTTGTCTTCT TGRAAAGCAC CAAGATTTGC TCTT flaB_UT2_R3 ACGCACTTGA 159 CTTGTCTTCT TGGAAAGCAC CYAAATTTGC TCTT Ft-G_UT_F ACCCAACTGA 160 ATGGAGCCTA AGCCATAAGC CCTTTCTCTA ACTTGT Ft-G_UT_R ACGCACTTGA 161 CTTGTCTTCA GCAATGACAA AGCTTGTTGA AAAAG glpQ_UT1_F1 ACCCAACTGA 162 ATGGAGCCCA GAACATACCT TAGAAKCTAA AGC glpQ_UT1_F2 ACCCAACTGA 163 ATGGAGCCAG AACATACATT AGAAGCCAAA GC glpQ_UT1_R1 ACGCACTTGA 164 CTTGTCTTCC CTTGTTGYTT ATGCCATAAK GGTT glpQ_UT1_R2 ACGCACTTGA 165 CTTGTCTTCC CTTGTTGTTT ATGCCAHAAG GGTT glpQ-Halp_UT2_F1 ACCCAACTGA 166 ATGGAGCCCA GAACATACCT TAGAAKCTAA AGC glpQ-Halp_UT2_F2 ACCCAACTGA 167 ATGGAGCCAG AACATACATT AGAAGCCAAA GC glpQ-Halp_UT2_R1 ACGCACTTGA 168 CTTGTCTTCC ACATTAGCAG AAATCAAATC AC glpO-Halp_UT2_R2 ACGCACTTGA 169 CTTGTCTTCG ATCAAATCTT TCGCTAAGRC TTAGTG glpQ-Halp_UT2_R3 ACGCACTTGA 170 CTTGTCTTCG ATCAAATCTT TCACTGAGAC TTAGTG glpQ-Halp_UT2_R4 ACGCACTTGA 171 CTTGTCTTCG ATCAAATCTT TCACTAAGGC TTAATG glpQ-Halp_UT2_R5 ACGCACTTGA 172 CTTGTCTTCG GGTATCCARG GTCCAAT H3N2_UT_F ACCCAACTGA 173 ATGGAGCAAG ACCAATYCTG TCACCTCTGA H3N2_UT_R ACGCACTTGA 174 CTTGTCTTCC AAAGCGTCTA CGCTGCAGTC C IGS1-Bunikis_UT1_F ACCCAACTGA 175 ATGGAGCGTA TGTTTAGTGA GGGGGGTG IGS1-Bunikis_UT1_R ACGCACTTGA 176 CTTGTCTTCG GATCATAGCT CAGGTGGTTA G IGS1-Bunikis_UT2_F ACCCAACTGA 177 ATGGAGCAGG GGGGTGAAGT CGTAACAAG IGS1-Bunikis_UT2_R ACGCACTTGA 178 CTTGTCTTCG TCTGATAAAC CTGAGGTCGG A IGS2-Derdakova_UT_F ACCCAACTGA 179 ATGGAGCCGA CCTTCTTCGC CTTAAAGC IGS2-Derdakova_UT_R ACGCACTTGA 180 CTTGTCTTCA GCTCTTATTC GCTGATGGTA IGS-5S-23S-Postic_UT_F ACCCAACTGA 181 ATGGAGCCTG CGAGTTCGCG GGAGA IGS-5S-23S-Postic_UT_R ACGCACTTGA 182 CTTGTCTTCT CCTAGGCATT CACCATA IGS-5S-23S-TK_UT_F ACCCAACTGA 183 ATGGAGCGAG TTCGCGGGAG AGTAGGTTAT TGCC IGS-5S-23S-TK_UT_R ACGCACTTGA 184 CTTGTCTTCT CAGGGTACTT AGATGKTTCA CTTCC IPC-gapDH_UT1_F ACCCAACTGA 185 ATGGAGCCCT GCCAAATATG ATGACATCAA G IPC-gapDH_UT1_R ACGCACTTGA 186 CTTGTCTTCG TGGTCGTTGA GGGCAATG ospA-Rudenko_UT_F ACCCAACTGA 187 ATGGAGCGAG CTTAAAGGAA CTTCTGATAA ospA-Rudenko_UT_R ACGCACTTGA 188 CTTGTCTTCG TATTGTTGTA CTGTAATTGT ospB_UT1_F ACCCAACTGA 189 ATGGAGCTGC GGTGACAGAA GACTC ospB_UT1_R ACGCACTTGA 190 CTTGTCTTCC AGCAGAAACT GTTAATTTTA CTTTACTC ospB_UT2_F ACCCAACTGA 191 ATGGAGCTGC GGTGACAGAA GACTC ospB_UT2_R ACGCACTTGA 192 CTTGTCTTCA ATCAGCAGAA ACTGTTAATT TTACTTTAC ospC-Bunikis_UT1_F ACCCAACTGA 193 ATGGAGCATG AAAAAGAATA CATTAAGTGC ospC-Bunikis_UT1_R ACGCACTTGA 194 CTTGTCTTCA TTAATCTTAT AATATTGATT TTAATTAAGG ospC-Bunikis_UT2_F ACCCAACTGA 195 ATGGAGCTAT TAATGACTTT ATTTTTATTT ATATCT ospC-Bunikis_UT2_R ACGCACTTGA 196 CTTGTCTTCT TGATTTTAAT TAAGGTTTTT TTGG ospC-Wang_UT1_F ACCCAACTGA 197 ATGGAGCAAA GAATACATTA AGTGCGATAT T ospC-Wang_UT1_R ACGCACTTGA 198 CTTGTCTTCG GGCTTGTAAG CTCTTTAACT ospD_UT1_F ACCCAACTGA 199 ATGGAGCGAG CTTAAAGGAA CTTCTGATAA ospD_UT1_R ACGCACTTGA 200 CTTGTCTTCG TATTGTTGTA CTGTAATTGT p66_UT1_F ACCCAACTGA 201 ATGGAGCGAG CTTAAAGGAA CTTCTGATAA p66_UT1_R ACGCACTTGA 202 CTTGTCTTCG TATTGTTGTA CTGTAATTGT p66_UT2_F ACCCAACTGA 203 ATGGAGCGAG CTTAAAGGAA CTTCTGATAA p66_UT2_R ACGCACTTGA 204 CTTGTCTTCG TATTGTTGTA CTGTAATTGT p66-Bunikis_UT1_F ACCCAACTGA 205 ATGGAGCGAT TTTTCTATAT TTGGACACAT p66-Bunikis_UT1_R ACGCACTTGA 206 CTTGTCTTCT GTAAATCTTA TTAGTTTTTC AAG p66-Bunikis_UT2_F ACCCAACTGA 207 ATGGAGCCAA AAAAGAAACA CCCTCAGATC C p66-Bunikis_UT2_R ACGCACTTGA 208 CTTGTCTTCC CTGTTTTTAA ATAAATTTTT GTAGCATC p66-Rudenko_UT1_F ACCCAACTGA 209 ATGGAGCCGA AGATACTAAA TCTGT p66-Rudenko_UT1_R ACGCACTTGA 210 CTTGTCTTCG CTGCTTTTGA GATGTGTCC par_A_UT1_F ACCCAACTGA 211 ATGGAGCGAG CTTAAAGGAA CTTCTGATAA par_A_UT1_R ACGCACTTGA 212 CTTGTCTTCG TATTGTTGTA CTGTAATTGT Yp3a_UT_F ACCCAACTGA 213 ATGGAGCCAT TGGACGGCAT CACGAT Yp3a_UT_R ACGCACTTGA 214 CTTGTCTTCA GTTGGCCAGC GATTCGA Yppla_UT_F ACCCAACTGA 215 ATGGAGCGAA AGGAGTGCGG GTAATAGGTT Yppla_UT_R ACGCACTTGA 216 CTTGTCTTCG GCCTGCAAGT CCAATATATG G

TABLE 3 Additional primers for LymeSeq Lyme Disease Next-Generation Sequencing Diagnostic Assay with the universal tail targets ACCCAACTGAATGGAGC (SEQ ID NO: 217) or ACGCACTTGACTTGTCTTC (SEQ ID NO: 218) SEQ Target Target Target Gene/ LIT Assay ID Purpose Taxon Region Type Assay Primer Sequence with UT NO: Species Borrelia 16S-set of 5 Sequence- I6S-1_UT 16S-1_UT_F ACCCAACTGAATGG 315 ID spp. assays to cover based AGCCGGGTGAGTA whole gene ACGCGTGGAT 16S-1_UT_R ACGCACTTGACTTG 316 TCTTCCCTCTCAGG CCGGTTACTTATC 16S-2_UT 16S-2_UT_F ACCCAACTGAATGG 317 AGCCGGTCACACTG GAACTGAGA 16S-2_UT_R ACGCACTTGACTTG 318 TCTTCGCTGCTGGC ACGT AATTAGC 16S-3_UT 16S-3_UT_F ACCCAACTGAATGG 319 AGCGCGAGCGTTGT TCGGGAT I6S-3_UT_R ACGCACTTGACTTG 320 TCTTCACTCAGCGT CAGTCTTGACC 16S-4_UT 16S-4_UT_F ACCCAACTGAATGG 321 AGCCGCTGTAAACG ATGCACACTTG 16S-4_UT_R ACGCACTTGACTTG 322 TCTTCACAACCATG CAGCACCTGTA 16S-5_UT 16S-5_UT_F ACCCAACTGAATGG 323 AGCGCAACGAGCG CAACCCTT 16S-5_UT_R ACGCACTTGACTTG 324 TCTTCTACAAGGCC CGAGAACGTATTCA C Species Borrelia IGS2 5S-23S Sequence- IGS2-5S-23S- IGS-5S-23S- ACCCAACTGAATGG 325 ID spp. based Postic_UT Postic_UT_F AGCCTGCGAGTTCG CGGGAGA IGS-5S-23S- ACGCACTTGACTTG 326 Postic_UT_R TCTTCTCCTAGGCA TTCACCATA Species Borrelia IGS rrs-rrlA rrs-rrlA_UT1 rrs- ACCCAACTGAATGG 327 ID spp. 16S-23S IGS rrlA_UT1_F1 AGCGGGTTCGAGTC CCTYAACCT new rrs- ACCCAACTGAATGG 328 assays rrlA_UT1_F2 AGCTTGGTTTAGAG 062216 CATCGGCTTTGC rrs- ACGCACTTGACTTG 329 rrlA_UT1_R1 TCTTCCCTTGCACT TTAGCGAAACAAC rrs- ACGCACTTGACTTG 330 rrlA_UT1_R2 TCTTCCCTTGTGCTT TAGTGAAACAAC rrs- ACGCACTTGACTTG 331 rrlA_UT1_R3 TCTTCACTTGCCAT ACGTAAACAACCGT rrs- ACGCACTTGACTTG 332 rrlA_UT1_R4 TCTTCCTCATGACT TGTCACACGTAAAC AAC rrs- ACGCACTTGACTTG 333 rrlA_UT1_R5 TCTTCGTTCAACTC CTCCTGGTCCCAA rrs- ACGCACTTGACTTG 334 rrlA_UT1_R6 TCTTCATCCTATAG ATGCAATCTCTTGW CC rrs- ACGCACTTGACTTG 335 rrlA_UT1_R7 TCTTCTTTGCATGT AATCAAGTCTTGGA ATTC rrs- ACGCACTTGACTTG 336 rrlA_UT1_R8 TCTTCTACTTTCAC CTCTAGACATTCTT GT rrs- ACGCACTTGACTTG 337 rrlA_UT1_R9 TCTTCTAGGTTGAT TCATGATCAGGTCC TT rrs- ACGCACTTGACTTG 338 rrlA_UT1_R1 TCTTCCGATTCGGT 0 CACGGCTCTTAC rrs- ACGCACTTGACTTG 339 rrlA_UT1_R1 TCTTCCCTTATGAT 1 TTAGTAACACAACG TAAGT rrs- ACGCACTTGACTTG 340 rrlA_UT1_R1 TCTTCAAGCTAGTA 2 ATGAATGTGGGATG TT Species Borrelia flaB Sequence- flaB_UT1 flaB_UT1_F ACCCAACTGAATGG 341 ID spp. based AGCGCWTCTGATG ATGCTGCTGGIA flaB_UT1_R1 ACGCACTTGACTTG 342 TCTTCGCATTCCAA GYTCTTCAGCTGT flaB_UT1_R2 ACGCACTTGACTTG 343 TCTTCGCATTCCAA GCTCTTCAGCWGT flaB_UT2 flaB_UT2_F1 ACCCAACTGAATGG 344 AGCACACCAGCRTC RCTTTCAGG flaB_UT2_F2 ACCCAACTGAATGG 345 AGCACACCAGCATC AYTAKCTGGA flaB_UT2_F3 ACCCAACTGAATGG 346 AGCACACCAGCATC ATTRGCTGGA flaB_UT2_R1 ACGCACTTGACTTG 347 TCTTCTTGGAAAGC ACCTAAATTTGCYC TT flaB_UT2_R2 ACGCACTTGACTTG 348 TCTTCTTGRAAAGC ACCAAGATTTGCTC TT flaB_UT2_R3 ACGCACTTGACTTG 349 TCTTCTTGGAAAGC ACCYAAATTTGCTC TT Species non- glpQ Sequence- glpQ_UT1 glpQ_UT1_F1 ACCCAACTGAATGG 350 ID Burgdorferi based AGCCCAGAACATA Borrelia CCTTAGAAKCTAAA spp. GC glpQ_UT1_F2 ACCCAACTGAATGG 351 AGCCAGAACATAC ATTAGAAGCCAAA GC glpQ_UT1_R1 ACGCACTTGACTTG 352 TCTTCCCTTGTTGY TTATGCCATAAKGG TT glpO_UT1_R2 ACGCACTTGACTTG 353 TCTTCCCTTGTTGTT TATGCCAHAAGGGT T Species B. bbk32 presence/ bbk32_UT bbk32_UT_F1 ACCCAACTGAATGG 354 ID burgdorferi absence AGCTGGAGGAGMC ss TATTGAAAGYAATG bbk32_UT_F2 ACCCAACTGAATGG 355 AGCTGAAGGAKAC TATTGAAAGYAATG bbk32_UT_R1 ACGCACTTGACTTG 356 TCTTCGCGTGTAGA ATACATTTGGGTTA GC bbk32_UT_R2 ACGCACTTGACTTG 357 TCTTCGACGTGTAG AATACATTTGGGTT TGC Species B. dbpA presence/ dbpA_UT2 dbpA_UT2_F1 ACCCAACTGAATGG 358 ID burgdorferi absence AGCCAGCCGCATCT GTAACTG dbpA_UT2_F2 ACCCAACTGAATGG 359 AGCTCAGTTCCCAT TGAAACTG dbpA_UT2_F3 ACCCAACTGAATGG 360 AGCTTYAGCYGCAT CTGAGAC dbpA_UT2_F4 ACCCAACTGAATGG 361 AGCTTCAGCTGCC WTTGAGAC dbpA_UT2_R ACGCACTTGACTTG 362 I TCTTCCAGGYAGCA AGGTATCAGA dbpA_UT2_R ACGCACTTGACTTG 363 2 TCTTCCRGGTAGYG GGGTATCAGA dbpA_UT2_R ACGCACTTGACTTG 364 3 TCTTCAACAGGTRG AAAGGYAGCA Species B. dbpB presence/ dbpB_UT2 dbpB_UT2_F1 ACCCAACTGAATGG 365 ID burgdorferi absence AGCCGCAAGCAAT CTTTCAGYTGTGT dbpB_UT2_F2 ACCCAACTGAATGG 366 AGCCTCAACCAATC TTTCAGCYGTGT dbpB_UT2_F3 ACCCAACTGAATGG 367 AGCCTTCAAGCAAT CTTTCACATGTGT dbpB_UT2_F4 ACCCAACTGAATGG 368 AGCCCTCAATTAAT CTTTCAGATGTGCT dbpB_UT2_F5 ACCCAACTGAATGG 369 AGCTTCAAGCAATC TTTCGGCTGTGT dbpB_UT2_F6 ACCCAACTGAATGG 370 AGCCTCCATTACTC TTTCGGCTGTGT dbpB_UT2_R ACGCACTTGACTTG 371 1 TCTTCRYAGCKCTT GAATCRTCYTYTAA GG dbpB_UT2_R ACGCACTTGACTTG 372 2 TCTTCAAGCAATGC TTGAATCSTMTTCT GA dbpB_UT2_R ACGCACTTGACTTG 373 3 TCTTCAAGCAAAGC TTGAATCGTCTTCC Species Anaplasma msp2 (major Ana- Ana- ACCCAACTGAATGG 374 ID phagocyto- surface protein) msp2_UT2 msp2_UT2_F AGCGGGAGAGTAA philum CGGAGARACWAAG G Ana- ACGCACTTGACTTG 375 msp2_UT2_R TCTTCCTGGCACCA 1 CCAATACCATAACC Ana- ACGCACTTGACTTG 376 msp2_UT2_R TCTTCCTGGCACCA 2 CCAATACCRTACC Species Ehrlichia 16S Sequence- Ehrl-16S_UT Ehrl- ACCCAACTGAATGG 377 ID genus based 16S_UT_F AGCGAGGATTTTAT CTTTGTATTGTAGC TAAC Ehrl- ACGCACTTGACTTG 378 16S_UT_R TCTTCTGTAAGGTC CAGCCGAACTGACT Species Ehrlichia 16S Sequence- Ehrl- Ehrl- ACCCAACTGAATGG 379 ID genus based 16S_UT2 16S_UT2_F AGCCAGGATTAGAT ACCCTGGTAGTCCA Ehrl- ACGCACTTGACTTG 380 16S_UT2_R TCTTCACGACACGA GCTGACGACA Species Ehrlichia sodB presence/ Ehrl- Ehrl- ACCCAACTGAATGG 381 ID genus absence sodB_UT sodB_UT_F AGCTTTAATAATGC TGGTCAAGTATGGA ATCAT Ehrl- ACGCACTTGACTTG 382 sodB_UT_R TCTTCAAGCRTGYT CCCATACATCCATA G Species B. ospB presence/ ospB_UT3 ospB_UT3_F1 ACCCAACTGAATGG 383 ID burgdorferi absence AGCGTYGAACTTAA AGGAACTTCCGAT ospB_UT3_F2 ACCCAACTGAATGG 384 AGCNTTGAGCTWA AAGGAACWTCTGA T ospB_UT3_F3 ACCCAACTGAATGG 385 AGCGTTGAGCTTAA AGGRGTTKCTGA ospB_UT3_F4 ACCCAACTGAATGG 386 AGCGGTGAGCTTAA AGGGGATTTTGA ospB_UT3_F5 ACCCAACTGAATGG 387 AGCGTTGAGCTTAA AGGCCTTTCTGAG ospB_UT3_R1 ACGCACTTGACTTG 388 TCTTCCCGMCTMCA AGACTTCCTTCA ospB_UT3_R2 ACGCACTTGACTTG 389 TCTTCCCGCCTACA AGATTTCCTGGA ospB_UT3_R3 ACGCACTTGACTTG 390 TCTTCCCACCAACA AGACTTCCTTCTAG T ospB_UT3_R4 ACGCACTTGACTTG 391 TCTTCCCACCAACT AGACTTCCTTTAAA C ospB_UT3_R5 ACGCACTTGACTTG 392 TCTTCCCACCAACA AGATTTCCTTCGAA C ospB_UT3_R6 ACGCACTTGACTTG 393 TCTTCCATTAGCTA CTTTTCCTTCAAGA G ospB_UT3_R7 ACGCACTTGACTTG 394 TCTTCCATTAGCTA GAGTTCCTTCAAGA G ospB_UT3_R8 ACGCACTTGACTTG 395 TCTTCTCAGCAGYT AGAGTTCCTTCAAG A Species B. ospC-TG presence/ ospC- ospC- ACCCAACTGAATGG 396 ID burgdorferi absence TG_UT1 TG_UT1_F AGCTCAGGRAAAG ATGGGAATRCATCT GC ospC- ACGCACTTGACTTG 397 TG_UT1_R TCTTCGRCTTGTAA GCTCTTTAACTGMA TTAG Species B. p66 presence/ p66_UT3 p66_UT3_F1 ACCCAACTGAATGG 398 ID burgdorferi absence AGCGCCYATGACY GGATTCAAA p66_UT3_F2 ACCCAACTGAATGG 399 AGCTTYGCACCTAT GACTGGRTTT p66_UT3_R ACGCACTTGACTTG 400 TCTTCGGYTTCCAT GTTGCTTGAAY p66_UT4 p66_UT4_F1 ACCCAACTGAATGG 401 AGCTGARGCTATCC ATCCAAGRCC p66_UT4_F2 ACCCAACTGAATGG 402 AGCGAAGCTGTCCA TCCAAGATTAG p66_UT4_R1 ACGCACTTGACTTG 403 TCTTCCGGTTTAGC TTGGAATACAGATG A p66_UT4_R2 ACGCACTTGACTTG 404 TCTTCCGGTTTTGC CTGGAATAAAGAT GA p66_UT4_R3 ACGCACTTGACTTG 405 TCTTCGGCYTAGCT TGGAAYATAGATG A p66_UT5 p66_UT5_F ACCCAACTGAATGG 406 AGCGCAATMGGAA AYTCAACATTC p66_UT5_R ACGCACTTGACTTG 407 TCTTCCRCTTGCAA ATGGGTCTATTCCT Species B. ospA ospA ospA_UT1 ospA_UT1_F1 ACCCAACTGAATGG 408 ID burgdorferi AGCGGITCTGGAAY ACTTGAAGG ospA_UT1_F2 ACCCAACTGAATGG 409 AGCGGATCTGGRRT RCTTGAAGG ospA_UT1_F3 ACCCAACTGAATGG 410 AGCGGTTCTGGAAS CCTTGARGG ospA_UT1_F4 ACCCAACTGAATGG 411 AGCGGRYCTGGGG TRCTTGAAGG ospA_UT1_F5 ACCCAACTGAATGG 412 AGCGGATCTGGGG GAAAGCTTGAAG ospA_UT1_F6 ACCCAACTGAATGG 413 AGCGGTTCTGGDGT RCTKGAAGG ospA_UT1_F7 ACCCAACTGAATGG 414 AGCGGATCTGGMW HGCYYGAAGG ospA_UT1_F8 ACCCAACTGAATGG 415 AGCGGMGCTGGAM AWCTTGAAGG ospA_UT1_R1 ACGCACTTGACTTG 416 TCTTCCAAGTYTGK TKCCRTTTKCTCTT G ospA_UT1_R2 ACGCACTTGACTTG 417 TCTTCCAAGYYTGG TWCCGTYTGCTCTT R ospA_UT1_R3 ACGCACTTGACTTG 418 TCTTCCMAGTGTAG TYCCGYTTGDTCTT G ospA_UT1_R4 ACGCACTTGACTTG 419 TCTTCCAAGTMTKG WWCCRTTTGCTCTT R ospA_UT1_R5 ACGCACTTGACTTG 420 TCTTCCAAGKGTAG TTTCGTTTKCTCTTG ospA_UT1_R6 ACGCACTTGACTTG 421 TCTTCCAAKTGTAG TATYRTTTGATCTT G ospA_UT1_R7 ACGCACTTGACTTG 422 TCTTCCAAGMKTRG TKCCGTTTGCTCTT G ospA_UT1_R8 ACGCACTTGACTTG 423 TCTTCCAAGTCTGG TTCCGTCTTTTCTTG ospA_UT1_R9 ACGCACTTGACTTG 424 TCTTCCAAGTGGTG TTCCGTTTGTTCTTG ospA_UT1_R1 ACGCACTTGACTTG 425 0 TCTTCCAAGTCTAT TTCCATTTGCTCTT G ospA_UT1_R1 ACGCACTTGACTTG 426 1 TCTTCCAAGTCTGG TTCCGTTAYCTCTT A ospA_UT1_R1 ACGCACTTGACTTG 427 2 TCTTCCAAGTCTGG TTCCATTTGCCCTT A Species Borrelia porin gene presence/ p66- p66_UT2_F ACCCAACTGAATGG 428 ID burgdorferi absence borrelia_UT2 AGCTGTAATTGCAG AAACACCTTTTGA p66_UT2_R ACGCACTTGACTTG 429 TCTTCGCTGCTTTT GAGATGTGTCC Genus Bartonella ssrA presence/ Bart- Bart- ACCCAACTGAATGG 430 ID absence ssrA_UT1 ssrA_UT1_F AGCGGCTAAATIAG TAGTTGCAAAYGAC A Bart- ACGCACTTGACTTG 431 ssrA_UT1_R TCTTCGCTTCTGTT GCCAGGTG Genus Babesia 18S sequence- Babe- Babe- ACCCAACTGAATGG 432 ID based 188_UT1 18S_UT1_F AGCACCGTCCAAA GCTGATAGGTC Babe- ACGCACTTGACTTG 433 18S_UT1_R TCTTCCGAAACTGC GAATGGCTCATTA Genus Rickettsia ompA presence/ Rkttsia- Rkttsia- ACCCAACTGAATGG 434 ID absence ompA_UT1 ompA_UT1_F AGCGGCATTTACTT ACRGTGSTGAT Rkttsia- ACGCACTTGACTTG 435 ompA_UT1_R TCTTCCCATGATTT GCAGCAAYAGCAT Rkttsia- Rkttsia- ACCCAACTGAATGG 436 ompA_UT2 ompA_UT2_F AGCCGYTAGCTGG GCTTAGRTATTC Rkttsia- ACGCACTTGACTTG 437 ompA_UT2_R TCTTCCGCCGRAAC TTTATTCTTGAATG Rkttsia- Rkttsia- ACCCAACTGAATGG 438 ompA_UT3 ompA_UT3_F AGCACTTAYGGTGG TGATTATAYTATC Rkttsia- ACGCACTTGACTTG 439 ompA_UT3_R TCTTCTGCAGCAAC AGCATTAKTACYG Rkttsia- Rkttsia- ACCCAACTGAATGG 440 ompA_UT4 ompA_UT4_F AGCGCTGRAGGAG 1 TAGCTAATGGT Rkttsia- ACCCAACTGAATGG 441 ompA_UT4_F AGCGCAGCAGGAG 2 TAGCTGATGAT Rkttsia- ACGCACTTGACTTG 442 ompA_UT4_R TCTTCMCGCAGCAG TACCGGTTAAAG Rkttsia- Rkttsia- ACCCAACTGAATGG 443 ompA_UT5 ompA_UT5_F AGCCAACCGCAGC RWTAATGCTAAC Rkttsia- ACGCACTTGACTTG 444 ompA_UTS_R TCTTCCCTCCCGTA TCTACCACTGAAC Rkttsia- Rkttsia- ACCCAACTGAATGG 445 ompA_UT6 ompA_UT6_F AGCTGCAGGAGCA GATAATGGTA Rkttsia- ACGCACTTGACTTG 446 ompA_UT6_R TCTTCGCCGGCAGT AATAGTAACAG Rkttsia- Rkttsia- ACCCAACTGAATGG 447 ompA_UT7 ompA_UT7_F AGCGGTGCAAGCC 1 AAGTAACATATAC Rkttsia- ACCCAACTGAATGG 448 ompA_UT7_F AGCAGGTACAAAT 2 CAAGTAACATATAC C Rkttsia- ACGCACTTGACTTG 449 ompA_UT7_R TCTTCAAACCGCCT 1 TCCGTTTCTG Rkttsia- ACGCACTTGACTTG 450 ompA_UT7_R TCTTCAATCCACCT 2 GCCGCTTCTG Genus Powassan presence/ Powass_UT Powass_UT_F ACCCAACTGAATGG 451 ID and deer absence I AGCGGCDGTAGGY tick viruses CATGTTTATGAC Powass_UT_F ACCCAACTGAATGG 452 2 AGCAGCTGTGGGCC ACGTCTATGAC Powass_UT_R ACGCACTTGACTTG 453 1 TCTTCCCGAAGGCA GGTGATCTTTG Powass_UT_R ACGCACTTGACTTG 454 2 TCTTCCAGAAGGCA GGTGGTCCTTG Internal Human gapDH presence/ IPC- IPC- ACCCAACTGAATGG 455 control absence gapDH_UT1 gapDH_UT1 AGCCCTGCCAAATA F TGATGACATCAAG IPC- ACGCACTTGACTTG 456 gapDH_UT1 TCTTCGTGGTCGTT R GAGGGCAATG Differen- Enterovirus VP1 presence/ EV-D68_UT EV- ACCCAACTGAATGG 457 tial strain D68 absence D68_UT_F1 AGCACCAGARGAA diagnos- GCCATACAAAC tics EV- ACCCAACTGAATGG 458 D68_UT_F2 AGCTGACACTTCAA GCAATGTTCGTA EV- ACCCAACTGAATGG 459 D68_UT_F3 AGCAACGCCGAAC TTGGTGTG EV- ACCCAACTGAATGG 460 D68_UT_F4 AGCAACACCGAAC CAGAGGAAG EV- ACGCACTTGACTTG 461 D68_UT_R1 TCTTCTGACACTTC AAGCAATGTTCGTA EV- ACGCACTTGACTTG 462 D68_UT_R2 TCTTCAACGCCGAA CTTGGTGTG EV- ACGCACTTGACTTG 463 D68_UT_R3 TCTTCAACACCGAA CCAGAGGAAG EV- ACGCACTTGACTTG 464 D68_UT_R4 TCTTCSCTGAYTGC CARTGGAATGAA EV- ACGCACTTGACTTG 465 D68_UT_R5 TCTTCATGTGCTGT TATTGCTACCTACT G Differen- Staphylococcus Sa_M4_UT2 Sa_M4_UT2 ACCCAACTGAATGG 466 tial aureus F AGCTAGCGTTGGTA diagnos- TTAAGTGGTTGT tics Sa_M4_UT2 ACGCACTTGACTTG 467 R TCTTCTCAAATCCA GCAAAGCCATCA Differen- Influenza A matrix gene presence/ H3N2_UT H3N2_UT_F ACCCAACTGAATGG 468 tial absence AGCAAGACCAATY diagnos- CTGTCACCTCTGA tics RNA target H3N2_UT_R ACGCACTTGACTTG 469 TCTTCTAGCGTTGG TATTAAGTGGTTGT Differen- Yersinia plasmid Yppla_UT Yppla_UT_F ACCCAACTGAATGG 470 tial pestis AGCGAAAGGAGTG diagnos- CGGGTAATAGGTT tics Yppla_UT_R ACGCACTTGACTTG 471 TCTTCAAGACCAAT YCTGTCACCTCTGA chromosome Yp3a_UT Yp3a_UT_F ACCCAACTGAATGG 472 AGCCATTGGACGGC ATCACGAT Yp3a_UT_R ACGCACTTGACTTG 473 TCTTCGAAAGGAGT GCGGGTAATAGGTT Differen- Francisclla SNP Ft-G_UT Ft-G_UT_F ACCCAACTGAATGG 474 tial tularemis AGCCTAAGCCATAA diagnos- GCCCTTTCTCTAAC tics TTGT Ft-G_UT_R ACGCACTTGACTTG 475 TCTTCCATTGGACG GCATCACGAT 

We claim:
 1. A kit for detection of at least one Borrelia species causing Lyme Disease or tick-borne relapsing fever (TBRF), the kit comprising: primer pairs targeting at least one region of Borrelia 16S rRNA and at least one region of flaB, ospA, ospB, ospC, glpQ, 16S-23S intergenic spacer (IGS1), 5S-23S intergenic spacer (IGS2), bbk32, dbpA, dbpB, and/or p66.
 2. The kit of claim 1, wherein primer pairs targeting the least one region of Borrelia 16S rRNA comprises sequences selected from the group consisting of: SEQ ID NOS: 1-10.
 3. The kit of claim 2, wherein the primer pairs targeting at least one region of flaB, ospA, ospB, ospC, glpQ, 16S-23S intergenic spacer (IGS), 5S-23S intergenic spacer (IGS2), bbk32, dbpA, dbpB, and/or p66 contain sequences selected from the group consisting of SEQ ID NOS: 11-48, SEQ ID NOS: 60-77, SEQ ID NOS: 97-100, and SEQ ID NOS: 219-293.
 4. The kit of claim 3, further comprises primer pairs containing sequences selected from the group consisting of SEQ ID NOS: 49-59, SEQ ID NOS: 78-96, SEQ ID NOS: 105-108, and SEQ ID NOS: 294-314.
 5. The kit of claim 4, further comprising a nucleotide polymerase, buffer, diluent, and/or excipien one or more primers comprising a sequence selected from SEQ ID NOS: 109 and 110 for amplifying human GAPDH as an internal control.
 6. The kit of claim 1, wherein the primer pairs are labeled
 7. The kit of claim 6, wherein the labeled primer pairs comprise a universal tail sequence.
 8. The kit of claim 6, wherein the labeled primer pairs comprise chain termination bases is labeled with a fluorescent label of a different wavelength that allows the sequencing to be performed in a single reaction.
 9. A kit for detecting one or more Borrelia species causing Lyme Disease or tick-borne relapsing fever (TBRF) within a sample from a subject, the kit comprising: a) primers targeting: at least one region of Borrelia 16S rRNA; at least one region of a 16S-23S intergenic spacer (IGS1); at least one region of a 5S-23S intergenic spacer (IGS2); at least one region of a flagella subunit B (flaB) gene; at least one region of a bbk32 gene; at least one region of a dbpA gene; at least one region of a dbpB gene; at least one region of an ospA gene at least one region of an ospB gene; at least one region of an ospC gene; at least one region of a p66 porin gene; and at least one region of a glpQ gene.
 10. The kit of claim 9, wherein the at least one region of Borrelia 16S rRNA contain sequences selected from the group consisting of: SEQ ID NOS: 1-10.
 11. The kit of claim 10, wherein: the at least one region of the 16S-23 S intergenic spacer (IGS1) contains sequences selected from the group consisting of SEQ ID NOS: 17-20 and 219-232; the at least one region of the 5S-23S intergenic spacer (IGS2) contains sequences selected from the group consisting of SEQ ID NOS: 11-16; the at least one region of the flagella subunit B (flaB) gene contains sequences selected from the group consisting of SEQ ID NOS: 21-29; the at least one region of the bbk32 gene contains sequences selected from the group consisting of SEQ ID NOS: 41-44; the at least one region of the dbpA gene contains sequences selected from the group consisting of SEQ ID NOS: 45-46 and 233-239; the at least one region of the dbpB gene contains sequences selected from the group consisting of SEQ ID NOS: 47-48 and 240-248; the at least one region of the ospA gene contains sequences selected from the group consisting of SEQ ID NOS: 274-293; the at least one region of the ospB gene contains sequences selected from the group consisting of SEQ ID NOS: 60-63 and 249-261; the at least one region of the ospC gene contains sequences selected from the group consisting of SEQ ID NOS: 64-69 and 262-263; the at least one region of the p66 porin gene contains sequences selected from the group consisting of SEQ ID NOS: 70-75 and 264-273; and the at least one region of the glpQ gene contains sequences selected from the group consisting of SEQ ID NOS: 30-40.
 12. The method of claim 9, wherein the amplification products are analyzed by size determination with agarose gel electrophoresis.
 13. The kit of claim 9, wherein the primer pairs comprise a universal tail sequence.
 14. The kit of claim 9, wherein the one or more Borrelia species are selected from the group consisting of: Borrelia afzelii, Borrelia americana, Borrelia andersonii, Borrelia anserina, Borrelia baltazardii, Borrelia bavariensis, Borrelia bissettii, Borrelia brasiliensis, Borrelia burgdorferi, Borrelia californiensis, Borrelia carolinensis, Borrelia caucasica, Borrelia coriaceae, Borrelia crocidurae, Borrelia dugesii, Borrelia duttonii, Borrelia garinii, Borrelia graingeri, Borrelia harveyi, Borrelia hermsii, Borrelia hispanica, Borrelia japonica, Borrelia kurtenbachii, Borrelia latyschewii, Borrelia lonestari, Borrelia lusitaniae, Borrelia mayonii, Borrelia mazzottii, Borrelia merionesi, Borrelia microti, Borrelia miyamotoi, Borrelia parkeri, Borrelia persica, Borrelia queenslandica, Borrelia recurrentis, Borrelia sinica, Borrelia spielmanii, Borrelia tanukii, Borrelia theileri, Borrelia tillae, Borrelia turcica, Borrelia turdi, Borrelia turicatae, Borrelia valaisiana, Borrelia venezuelensis, Borrelia vincentii, and Candidatus Borrelia texasensis.
 15. The kit of claim 9, wherein the one or more Borrelia species are selected from the group consisting of: Borrelia burgdorferi, Borrelia garinii, Borrelia mayonii, and Borrelia afzelii.
 16. The kit of claim 9, further comprising detecting in the sample a Babesia species, an Ehrlichia species, a Bartonella species, Francisella tularensis, Yersinia pestis, Staphylococcus aureus, Anaplasma phagocytophilum, Enterovirus, Powassan and deer tick virus, Rickettsia species, and/or Influenza by subjecting the DNA and/or RNA from the sample to a second PCR amplification reaction using primer pairs containing sequences selected from the group consisting of: SEQ ID NOS: 49-55 for detection of Anaplasma phagocytophilum; SEQ ID NOS: 56-59 for detection of an Ehrlichia species; SEQ ID NOS: 78-86 for detection of Enterovirus; SEQ ID NOS: 87-88 for detection of Staphylococcus aureus; SEQ ID NOS: 89-90 for detection of Influenza; SEQ ID NOS: 91-94 for detection of Yersinia pestis; SEQ ID NOS: 95-96 for detection of Francisella tularensis; SEQ ID NOS: 105-106 for detection of a Bartonella species; SEQ ID NOS: 107-108 for detection of a Babesia species; SEQ ID NOS: 294-310 for detection of a Rickettsia species; and SEQ ID NOS: 311-314 for detection of a Powassan and deer tick virus.
 17. The kit of claim 9, further comprises a nucleotide polymerase, buffer, diluent, and/or excipient; one or more primers comprising a sequence selected from SEQ ID NOS: 109 and 110 for amplifying human GAPDH as an internal control; and the primer pairs are labeled
 18. A method of detecting one or more Borrelia species causing Lyme Disease or tick-borne relapsing fever (TBRF) within a sample from a subject, the method comprising: a) subjecting DNA and/or RNA from the sample to a multiplex PCR amplification reaction with primers targeting: at least one region of Borrelia 16S rRNA; at least one region of a 16S-23S intergenic spacer (IGS1); at least one region of a 5S-23S intergenic spacer (IGS2); at least one region of a flagella subunit B (flaB) gene; at least one region of a bbk32 gene; at least one region of a dbpA gene; at least one region of a dbpB gene; at least one region of an ospA gene at least one region of an ospB gene; at least one region of an ospC gene; at least one region of a p66 porin gene; and at least one region of a glpQ gene; and b) analyzing amplification products resulting from the PCR amplification reaction to detect the one or more Borrelia species.
 19. The method of claim 18, wherein the amplification products are analyzed to determine the sequence of each amplification product.
 20. The method of claim 18, wherein the sequence of each amplification product is mapped to a reference library of known Borrelia sequences to detect the one or more Borrelia species and to identify Borrelia species that cause Lyme Disease and Borrelia species that do not cause Lyme Disease. 